Immunohistofluorescence Study of the Actions of Manganese on the β‐Arrestin and Gβγ Mechanisms of Dopamine D2 Signal Transduction Pathway in Crassostrea virginica
Authors: Eid Mohamed , Santos Rafael A. , Carroll Margaret A. , Catapane Edward J. 
Published: June 2021

Manganese (Mn) causes manganism, a Parkinson's‐like disease. Manganism disrupts dopamine (DA) neurotransmission, but the mechanism is not resolved. Gill lateral cell (GLC) cilia of Crassostrea virginica are innervated by serotonin and dopamine. DA slows cilia beating; serotonin speeds them up. Our lab showed DA receptors in GLC are D2‐type (D2R) and Mn disrupts the DA induced cilio‐inhibition. D2R are G protein‐coupled receptors. D2R activation splits the Gi/o protein into Gβγ and Gαi/o subunits. Gαi/o inhibits adenylyl cyclase. Gβγ activates phospholipase C (PLC), opens GIRK channels and closes L‐type Ca2+ channels. Continued activation of D2R results in β‐Arrestin binding, which desensitizes and targets them for internalization. β‐arrestin and Gβγ have not been well studied in bivalves and the effects of Mn on them is unknown. We hypothesize they can be visualized in oyster GLC by immunohistofluorescence, and if so, we will determine if Mn or DA affects their visualization. Briefly, gills were dissected, snap frozen, cryostat sectioned at 10 μm, fixed with EDAC, treated with blockers, and incubated with 1° antibodies for β‐Arrestin or for Gβγ, then FITC labelled 2° antibodies. Sections were viewed on a fluorescence microscope with a Leica DFC400 camera, 50 watt mercury lamps and FITC excitation/emission filters. All sections were photographed with the same camera setting. Results showed control sections had bright green fluorescence in cytoplasm and along the plasma membrane indicating the presence of β‐arrestin and Gβγ. In other experiments gills were treated for 1 or 24 hours with 500μM of Mn. Fluorescence intensity (FI) was quantified using ImageJ from NIH. Results showed compared to controls, Mn treatments decreased FI for both β‐arrestin and Gβγ about 10% in the 1 hour treatments and 15% in the 24 hour treatment. Zn, which was used as a comparison caused either no, or a less than 5% decrease in FI for both β‐arrestin and Gβγ. In other experiments gills were treated for 1 hour with DA or DA plus Mn. DA had no effect on the FI of β‐arrestin. However, DA treatment increased FI intensity of Gβγ by more than 25%, even when Mn was present. The study shows β‐Arrestin and Gβγ are present in GLC of C. virginica and Mn caused a small but significant reduction in their FI. DA treatments increased Gβγ FI, but not β‐arrestin FI. This study provides new knowledge of the actions of Mn on the D2R pathway in bivalve gill. Our future experiments will test if Mn negatively effects the physiological actions of β‐arrestin and Gβγ on lateral cell cilia activity.Support or Funding InformationThis work was supported grant 2R25GM06003 of the Bridge Program of NIGMS, NIH grant K12GM093854‐07A1 IRACDA Program of Rutgers University and 604060048 of PSC‐CUNY.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.