Salivary cystatins influence ingestion of capsaicin-containing diets in the rat.

Dietary capsaicin consumed by rats over several days induces cystatin-like substances in submandibular saliva. Yet the physiological role of these salivary proteins has not been thoroughly investigated. Salivary cystatins in the rat submandibular glands are known to be induced by chronic treatment with the sympathetic beta-agonist, isoproterenol. In the present study, the possible roles of the salivary proteins on food intake were examined by comparing consumption of a capsaicin-adulterated (0.05%) diet in rats with and without isoproterenol pretreatment (0.1 and 5.0 mg/kg, 5 days). Electrophoretic analysis performed prior to feeding trials revealed that the group pretreated with 5 mg/kg isoproterenol had large amounts of cystatin in the saliva compared with the group pretreated with 0.1 mg/kg isoproterenol and control group. The group treated with 5 mg/kg isoproterenol showed greater consumption of the capsaicin-adulterated diet than the other groups until the 3rd day of trials. Bilateral removal of the submandibular and sublingual glands neutralized the effects of isoproterenol. Induction of salivary cystatins by isoproterenol treatment was not mimicked by systemic and intragastric administration of capsaicin. These results suggest that cystatins are included in the salivary proteins induced by capsaicin and that they contribute to enhanced ingestion of the capsaicin diet. Induction of salivary cystatins may be triggered by irritation of the oral mucosa by capsaicin.

Behavioral taste similarities and differences among monosodium L-glutamate and glutamate receptor agonists in C57BL mice.

Monosodium L-glutamate (MSG) and 5'-ribonucleotides elicit umami taste in humans and probably in some species of animals. Previous studies suggest that taste-mGluR4 and NMDA receptor may be involved in taste transduction for umami, but behavioral responses in rats do not support the involvement of NMDA receptor. In the present study, behavioral similarities and differences among MSG, mGluR4 agonist L(+)-2-amino-4-phosphonobutyrate (L-AP4), and NMDA receptor agonist N-methyl-D-aspartate (NMDA) were compared in C57BL mice by using a conditioned taste aversion paradigm. Mice conditioned to avoid either MSG or 10 mM L-AP4 appeared to avoid MSG, disodium 5'-inosinate (IMP), a mixture of MSG and IMP, and L-AP4, but not NMDA. Aversive conditioning to either sucrose or NMDA was generalized only to a mixture of MSG+IMP or NaCl. However, aversive conditioning to L-AP4 at 1 mM was generalized to NMDA and the umami substances. Lick rates for L-AP4 increased by mixing with (RS)-alpha-cycloprophy-4-phosphonophenylglycine (mGluR4 antagonist) when animals were conditioned to avoid MSG or L-AP4. Lick rates for NMDA also either decreased or increased by mixing with glycine (NMDA receptor coagonist) or D(-)-2-amino-5-phosphonopentanoic acid (NMDA receptor antagonist) when animals were conditioned to avoid L-AP4 or NMDA. In sucrose-conditioned mice. gurmarin (a sweet inhibiting peptide) suppressed the avoidance of sucrose and a mixture of MSG and IMP, but not L-AP4 and NMDA. The results suggest the possibility that to C 57BL mice MSG may taste similar to L-AP4 but different from NMDA, although both types of glutamate receptors as well as gurmarin-sensitive sweet receptor may be involved in perception of umami taste.

Capsaicin induces cystatin S-like substances in submandibular saliva of the rat.

Irritating dietary substances such as tannin and papain have been reported to alter the morphology of salivary glands and their secretions. Such alterations can be one line of protection from toxic or irritating substances in food. We investigated the effects of dietary capsaicin (a pungent ingredient of hot red pepper) on the rat submandibular gland and its secretions. Several groups of animals were offered either control diets or diets containing capsaicin (from 0.0001 to 0.1%) for seven days. Higher concentrations suppressed food consumption for two days, after which only the highest concentration continued to reduce intake. The relative weight of the salivary glands in capsaicin-diet groups increased in a dose-dependent fashion, and new proteins appeared in the submandibular saliva. Chromatographic and electrophoretic properties of these proteins were identical or similar to those of isoproterenol-induced proteins. After affinity chromatography of the new protein fraction on a Cm-papain Sepharose 4B column, SDS-electrophoresis of the eluate revealed three major bands (15,500, 16,500, and 28,000 kDa). Hydrolysis of N-benzoyl-D,L-arginine-p-nitroanilide by papain (a cysteine protease) decreased in the presence of the new protein fraction, suggesting that these proteins have cystatin-like activity (inhibition of cysteine protease). Denervation of the glossopharyngeal nerve suppressed induction of these proteins. The results suggest that dietary capsaicin induces cystatin S-like substances in submandibular saliva by stimulating the reflex arc involving the glossopharyngeal nerve. These proteins likely facilitate ingestion of diets containing the irritating substance.

Induction of salivary gurmarin-binding proteins in rats fed gymnema-containing diets.

Gymnema sylvestre, a tropical plant, contains gurmarin that selectively suppresses sucrose responses of the chorda tympani nerve in rats and mice. We investigated preference for taste solutions and saliva composition in rats fed a diet containing this plant (gymnema diet). Preference for 0.01 M sucrose and a mixture of 0.03 M sucrose and 0.03 mM quinine-HCl significantly decreased at 1-2 days after the start of the gymnema diet and subsequently returned closely to the control levels within about a week. There was no significant change in preference for NaCl, monosodium glutamate and quinine-HCl during feeding trials. Submandibular saliva of rats fed the gymnema diet for 4 and 14 days showed an inhibitory effect on immunoreaction between gurmarin and antigurmarin serum. Analyses using electrophoresis and affinity chromatography indicated that the saliva contains gurmarin binding proteins with molecular weights of 15, 16, 45, 60 and 66 kDa. These results suggest that reduction of preference for sucrose was probably caused by gurmarin contained in the gymnema diet and subsequent restoration of the preference may be due to suppression of the effect of gurmarin by salivary gurmarin-binding proteins induced by the gymnema diet.

Enhanced responses of the chorda tympani nerve to nonsugar sweeteners in the diabetic db/db mouse.

Genetically diabetic db/db mice show greater neural and behavioral responses to sugars than lean control mice. The present study examined chorda tympani responses of db/db mice to nonsugar sweeteners and their inhibition by a sweet response inhibitor, gurmarin. The results showed that responses to sucrose, saccharin, glycine, L-alanine, and D-tryptophan, but not to D-phenylalanine, were approximately 1.5 times greater in db/db mice than in control mice. Treatment of the tongue with gurmarin suppressed responses to these sweeteners in db/db and control mice, but the extent of suppression was considerably smaller in db/db mice. The magnitudes of gurmarin-sensitive components of the response to sweeteners in db/db mice were not significantly different from those in control mice, whereas the magnitudes of gurmarin-insensitive components in db/db mice were about twice as large as those in control mice. These results suggest that the enhancement of chorda tympani responses in db/db mice to sucrose and other nonsugar sweeteners may occur through gurmarin-insensitive membrane components.

Glossopharyngeal denervation alters responses to nutrients and toxic substances.

Functional roles of the glossopharyngeal (GL) nerve on food and fluid intake were studied by examining effects of the GL denervation on two biologically different activities induced by specific diets using mice and rats. First, we examined whether GL section alters the acceptability of a bitter tasting essential amino acid, L-lysine (Lys), by Lys-deficiency in mice. The aversion threshold for Lys, normally about 3 uM in mice, increased to about 300 uM when mice were fed the Lys-deficient diet for 10 days. This increase of the Lys aversion threshold (increase of acceptability for Lys) by Lys-deficiency was also evident in mice with the chorda tympani denervation but was not observed in mice with the GL denervation. Next, we examined whether GL section alters the induction of a salivary protein, cystatin S (a cysteine proteinase inhibitor), by a diet containing papain (a cysteine proteinase) in rats. GL denervation largely inhibited the induction of cystatin S in the rat submandibular glands by papain. These results collectively suggest that chemosensory information conveyed by the GL nerve plays important roles on recognition of both nutrient and toxic compounds in the diet and induction of biological responses that protect the animal from both nutritional deficiency and exogenous toxic compounds.

Genetically variable taste sensitivity to D-amino acids in mice.

Behavioral and neural responses to D-amino acids were compared between two inbred strains, C57BL and BALB mice. In both strains, an aversion conditioned to D-valine, D-leucine, D-methionine, D-histidine or D-tryptophan generalized to sucrose, whereas an aversion to D-alanine or D-serine did not generalize to sucrose. Generalization patterns across various test stimuli for each of these 7 D-amino acids were significantly correlated between two strains. However, an aversion conditioned to D-phenylalanine generalized to sucrose in C57BL mice, but not in BALB mice. Application of a proteolytic enzyme, Pronase E, to the tongue reduced chorda tympani responses to sucrose and D-amino acids to which a conditioned aversion generalized to sucrose. Again, only in C57BL mice, Pronase inhibited D-phenylalanine responses. These comparable results indicate that sweet taste response is genetically highly variable only to D-phenylalanine among 8 D-amino acids tested.

Effects of parasympathectomy on androgen responses of the rat submandibular gland.

The effects of androgen on the activities of esteropeptidase and delta 4-3-ketosteroid 5 alpha-reductase were investigated. Ratios of the denervated side to the non-operated side for gland weight and esteropeptidase activity were higher in androgenized females than in untreated females. The specific activities of 5 alpha-reductase were increased by denervation but the total activities were decreased. The results suggest that parasympathectomy leads to higher androgen responsiveness in the rat submandibular gland, which relates to the observed reduction in weight loss of the gland and the increases in its esteropeptidase activities.

Alteration of salt taste sensitivity by the neonatal removal of sublingual glands in the rat.

1. Adult rats with the surgical removal of sublingual glands at their 10 days of age did not prefer NaCl solution of any concentration to water, whereas those with sham-operation or the removal of submandibular glands preferred 0.03 or 0.1 M NaCl to water. 2. Magnitudes of inhibition of chorda tympani responses to NaCl by the lingual treatment of 0.1 mM amiloride were greater in neonatally sublingual removed rats than in sham-operated or submandibular removed ones. 3. These results suggest that the removal of sublingual glands in neonatal period of the rat could increase the amount of the amiloride-sensitive Na receptor mechanism on the taste cell membrane in its adulthood.

Effects of repeated androgen treatments on metabolism and nuclear binding of androgen in the infant murine submandibular gland.

1. Androgen responsiveness of esteropeptidase of the murine submandibular gland developed rapidly in normal males compared with in normal females and castrated males. 2. Repeated treatments of infant mice of both sexes with testosterone (T), 5 alpha-dihydrotestosterone (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol increased androgen responsiveness of this enzyme, but did not affect those of 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDase; androgen metabolizing enzymes) of the gland. 3. Exchange assay of nuclear androgen receptor using 3H-DHT showed that in both sexes, amounts of binding in animals pretreated with T were higher than those in animals pretreated with sesame oil. 4. These results suggest that there is parallelism between the androgen responses and amounts of nuclear androgen binding, not androgen responses of 5 alpha-reductase and 3 alpha-HSDase.

Qualitative discrimination of gustatory stimuli in three different strains of mice.

Qualitative similarities and differences among various taste stimuli were examined by comparing the generalization patterns of a conditioned aversion from single chemicals to other compounds in 3 different strains of mice (BALB, C3H and C57BL mice). It was observed as a common characteristic in all 3 strains of mice that generalization gradients among sugars and saccharin Na appeared in the order sucrose--saccharin Na--fructose--glucose--maltose, in which the closer stimuli generalized more strongly to each other. Strain differences were found in sensitivities to D-phenylalanine and L-proline, which generalized to sugars and saccharin Na in C57BL mice, but not in BALB and C3H mice. These strain differences correspond quite well to those previously observed in the responses of single chorda tympani fibers to these amino acids in the 3 strains of mice. A hierarchical cluster analysis and a multidimensional scaling analysis showed that 15 compounds including the 4 basic taste stimuli (sucrose, NaCl, HCl and quinine-HCl) were classified into 7 different groups according to their behavioral similarities and some amino acids were not grouped with any of the 4 basic taste stimuli in the 3 strains of mice. These results suggest the possibility that mice perceive tastes of these amino acids in a way different from human taste primaries.

Gustatory neural responses in three different strains of mice.

Taste sensitivity in mice and its strain variation were studied by examining integrated responses and single fiber discharges of the chorda tympani nerve to various taste stimuli in the 3 different strains of mice (BALB, C3H and C57BL mice). A comparison among integrated responses of the 3 strains demonstrated that C57BL mice possess the highest sensitivity to sucrose and the lowest to acids, whereas the opposite is true for BALB mice. These 3 strains of mice commonly possess higher sensitivity to divalent chloride salts than to NaCl. Measures of the breadth of responsiveness showed that single fibers of all 3 strains of mice are relatively more narrowly tuned to taste stimuli than those of rats and hamsters, although the specificity of fibers tends to be greater in the order of C57BL greater than C3H greater than BALB mice. A cluster analysis of fibers demonstrated that two distinct fiber types, a sweet- and Na-type, commonly exist in all 3 strains of mice. The most clear strain difference was found in the sensitivity to D-phenylalanine, which produced good responses in 'sweet-type' fibers of C57BL mice but not in those of C3H and BALB mice. This suggests the possibility that D-phenylalanine has a taste to C57BL mice that is similar to that of sucrose and that it tastes different to C3H and BALB mice.

Effects of androgens on the activity of steroid-metabolizing enzymes in the murine submaxillary gland.

Effects of androgens on the activity of androgen-metabolizing enzymes have been investigated in the murine submaxillary gland. 5 alpha-reductase activity is androgen-dependent, and 17 beta-hydroxysteroid dehydrogenase activity is androgen-resistant. The level and androgen-responsiveness of 5 alpha-reductase in the murine submaxillary gland are not imprinted during the neonatal period by testicular androgens. Circulating androgen may have a cumulative effect on androgen response of 5 alpha-reductase.

Electrophoretic patterns of proteins from the dentary, vertebra and scale in the ayu fish, Plecoglossus altivelis, Temminck et Schlegel.

The electrophoretic patterns of the proteins extracted from dentary, vertebra and scale with the phosphate buffer, 0.1 NHcl and SDS solution were studied to investigate their molecular aspects. The banding patterns and relative amounts of the proteins from dentary, vertebra and scale showed a basic similarity.