Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction

Abstract

R3. R3, PLCβ2, and γ13 positive cells. receptor expressed in taste cells and our data suggest it plays an important role in bitter taste transduction.

Background

R3.

Results

R3, PLCβ2, and γ13 positive cells.

Conclusions

receptor expressed in taste cells and our data suggest it plays an important role in bitter taste transduction.

Background

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receptor isotype that is expressed in taste cells.

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].

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Receptor Isotypes

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R3. The scale bar in each figure represents 10 μm.

R3 with known bitter signaling components

receptor expressed in taste cells, and that it is found in the same subset of taste cells as other components known to be involved with bitter taste transduction.

R3 IR cells are α-gustducin IR.

R3 IR and PLCβ2 IR.

R3 IR and γ13 IR.

Discussion

R3 labeling. However, using this method with α-gustducin, PLCβ2, and γ13 antibodies did not alter their immunoreactivities; similar results were obtained with and without antigen retrieval. Thus, we do not believe that antigen retrieval compromised our interpretation of the results.

].

]. Further experiments will be required to identify the additional alpha subunits that couple to this pathway, and the receptors that activate these G proteins.

R3 may be involved with sweet as well as bitter taste transduction.

oscillations.

pathway in taste cells.

Conclusions

and cAMP dependent kinases, are consistent with known characteristics of bitter signaling in taste cells.

R3, α-gustducin, PLCβ2, and γ13 in circumvallate taste buds.

Animals

Adult male Sprague Dawley Rats and adult C57/B1 male or female mice were obtained from Charles River Laboratories (Wilmington, MA). Animals were cared for in compliance with the Colorado State University Animal Care and Use Committee.

Tissue preparation

Rats or mice were deeply anesthetized by intraperitoneal injections of Sodium Pentobarbitol, 40 mg/Kg (Veterinary Laboratories, Inc., Lenexa, KS). Following anesthetization animals were injected intracardially with heparin (Elkins-Sinn, Inc., Cherry Hill, NJ) and 1% sodium nitrate. Rats were perfused with 80 ml of 4% paraformaldehyde and mice with 30 ml. Following perfusion tongues were removed and immediately placed into fresh 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) in 0.1 M phosphate buffer for approximately twenty minutes. Tongues were then put into a 20% sucrose solution in 0.1 M phosphate buffer overnight for cryoprotection. Forty micron sections were cut on a Leitz 1729 digital Kryostat and collected in 0.1 M phosphate buffered saline (PBS, pH7.2). Following sectioning, the slices were washed in PBS three times for ten minutes each at room temperature. Antigen retrieval was performed by placing sections into a 10 mM sodium citrate solution at 80°C for 30 minutes. This was done to help disrupt protein cross-bridges formed by formalin fixation and expose antigen binding sites. In some experiments the incubation time in sodium citrate was reduced to 5 minutes, and labeling was still apparent. All sections were incubated in blocking solution for 1-2 hours at room temperature. Blocking solution contained 0.3% Triton X-100, 1% normal goat serum, and 1% bovine serum albumin in 0.1 M PBS. All chemicals were purchased from Sigma Chemical Corporation (St. Louis, MO) unless otherwise noted.

Antibodies

]. All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). These included: Rhodamine Red X anti-rabbit (# 111-295-045), Rhodamine Red X anti-mouse (# 115-295-146), Cy-5 anti-mouse (# 115-175-146), Flourescein (FITC) anti-rabbit (# 711-095-152). Cy-5 anti mouse antibodies were tested on mouse tissue prior to these experiments to insure no background labeling.

Single label Immunocytochemistry

R3 labeled sections, were incubated in Rhodamine anti-mouse. Following incubation with secondary antibodies, sections were washed three times for ten minutes in PBS and mounted on RITE-ON micro slides (Becton, Dickinson and Company, Portsmouth, NH) using Flouromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL, cat# 0100-01) and coverslipped (VWR Scientific, Media, PA). Slides were stored at 4°C.

Double Label Immunocytochemistry

R3 double-labeled sections, Cy-5 anti-mouse secondary antibodies were applied. Rabbit anti-α-gustducin was visualized using FITC anti-rabbit (1:100) secondary antibodies. Both rabbit anti-PLCβ2 and rabbit anti-γ13 were labeled with FITC anti rabbit (1:100). Incubations with secondary antibodies were done at room temperature for two hours. Sections were then washed in PBS three times for ten minutes each, mounted using flouromount-6, and coverslipped.

Imaging

Lingual sections were viewed with an Olympus Fluoview laser scanning confocal microscope. Sequential scanning techniques were used for some double-label experiments and showed no differences from simultaneous scans. There is no overlap between the excitation and emission spectra for the FITC and Cy5 secondary antibodies used in the double label experiments. Images were captured with an Olympus FVX-IHRT Fluoview Confocal Laser Scanning Microscope. Lasers included Argon 488 nm, HeNe 543 nm, and HeNe 622. Fluoview software was used for data acquisition. Images were processed and printed using Photoshop 6.0 software.

Acknowledgements

We thank Dr. Vince Dionne for the initial motivation for this study, and Ms. Barbel Böttger for the suggestion of using antigen retrieval. We also thank Dr. Kathryn Medler for technical advice and Dr. Tom Finger for comments on the manuscript. RFM is an Associate Investigator of the Howard Hughes Medical institute. This study was supported by NIH grants DC00244 and DC00766 to SCK and DC03155 to RFM.

26:259-265, 2001).