α-Gus activates a retinal PDE in vitro
]. If a similar activity occurs in taste cells, chronic absence of α-gus might result in altered cAMP levels. Hence, we measured total cAMP in CV taste bud-enriched epithelium from α-gus-/-
mice. We also carried out parallel measurements of cAMP levels in adjacent non-taste lingual epithelium as a control. The resting level of cAMP in α-gus+/+
circumvallate epithelium was 2.65 ± 0.35 (mean ± s.e.m.) pmole cAMP/μg protein. The value is similar to that measured from rat CV epithelium [14
]. The basal level of cAMP was approximately 3.8 fold higher in CV epithelium from α-gus-/-
mice as compared to α-gus+/+
, a highly significant difference (p=0.0005; n=6; t-test; ). We attributed this difference to taste buds because adjacent regions of non-taste epithelium had much lower resting levels of cAMP, and these did not differ between genotypes (p=0.504; n=6; t-test). The data suggest that cAMP levels in taste buds are controlled by a PDE that is primarily regulated by α-gus, and may be active in the absence of taste stimulation.
Taste buds in Gus-/- mice have elevated resting levels of cAMP
mice have elevated basal levels of cAMP, phosphorylation by PKA could also be chronically elevated and this could underlie the decreased bitter sensitivity observed in α-gus-/-
]. In that earlier study, it could not be determined whether residual responses to bitter tastants were in cells of the α-gus lineage. To resolve this uncertainty, we used mice in which GFP was expressed in α-gus lineage taste cells of α-gus-/-
mice (). Taste cells, loaded with fura-2, were stimulated with denatonium (10 mM). Denatonium elicited little or no increase in intracellular Ca2+
in the GFP-labeled taste cells of the α-gus-/-
mice. To determine if elevated cAMP and PKA-dependent phosphorylation were responsible for this loss of sensitivity, we also examined bitter responses in the presence of the membrane-permeant PKA inhibitor, H-89 (10 μM). After treatment with H-89, Ca2+
responses in GFP-labeled α-gus-/-
cells were larger (). Responses to denatonium were unmasked by H-89 in 7 of 21 GFP-labeled α-gus-/-
taste cells that were previously unresponsive to denatonium (e.g. ). On average, responses to denatonium in α-gus-/-
cells were significantly enhanced, 6.5-fold in the presence of H-89 (). We noted that H-89 caused a slight elevation of the baseline Ca2+
in many taste cells, even prior to taste stimulation. This suggests that both basal Ca2+
levels and taste-evoked Ca2+
signals are regulated by PKA activity.
The GFP label is present in taste cells of both Gus+/+ and Gus-/- mice
H-89 increases the magnitude of Ca2+ responses to denatonium in gustducin-lineage taste cells from Gus+/+ and Gus-/- mice
Next, we asked if H-89 had a similar enhancing effect on Ca2+ responses to denatonium in gus-expressing taste cells of α-gus+/+ mice. Surprisingly, we found that H-89 also significantly increased Ca2+ responses to denatonium in α-gus+/+ mice (2.9-fold; ). After H-89 treatment, responses to denatonium were unmasked in 5 of 26 previously unresponsive Gus-GFP-positive cells.
The effect of H-89 on individual taste cells was reversible and repeatable () in both knockout and wildtype mice. The vehicle, DMSO, had no effect on either resting Ca2+ level or denatonium responses (n=4; data not shown). Although 10μM H-89 is selective for PKA, it may also block PKC slightly. Thus, we applied a robust, membrane permeant inhibitor of PKC, bisindolymaleimide I (Bis) to GFP-labeled cells of α-gus+/+ mice. Bis (0.15 μM) had no effect on either resting Ca2+ levels or denatonium responses (; p=0.78; paired t-test; n=4), suggesting that the effect of H-89 on bitter responses arises from a specific block of PKA.
Ca2+ responses to 10mM denatonium were on average, 2.5-fold larger in GFP-labeled taste cells of gus+/+ mice relative to gus-/- mice (; a significant difference, p=0.0072; t-test with Bonferroni correction for 3 way comparison across genotypes, and for H-89 within each genotype). We infer that the depressed responses to denatonium in the α-gus-/- cells arise from the elevated basal levels of cAMP, which would constitutively stimulate PKA.