Upregulation of the Adenylyl Cyclase Pathway in Optic Nerve Head (ONH) Astrocytes From African American (AA) Donors: Pressure-Dependent Increase of Intracellular Adenosine 3’, 5’ Cyclic Monophosphate (cAMP)

Abstract

Purpose:: The membrane-bound adenylyl cyclase catalyzes the conversion of ATP into cAMP and represents a major family of effector enzymes for G protein-coupled receptors. Our recent microarray analyses demonstrated upregulation of adenylyl cyclases , ADCY3 and ADCY9, in primary cultures of ONH astrocytes from AA compared to Caucasian Americans(CA) donors. Because intracellular cAMP levels have profound effects on astrocyte functions, we determined the effects of hydrostatic pressure (HP) on cAMP levels in normal ONH astrocytes from CA and AA age-matched donors.

Methods:: Primary ONH astrocytes cultured from 6 AA and 6 CA normal donors were used to perform cAMP assays using EIA methods. Cells were divided into four groups: basal serum free (BSF) conditions, 500µM IBMX, IBMX+HP 15’ and IBMX+ HP 30’respectively. All experiments were repeated in triplicate. Real-time PCR was used to determine mRNA levels for ADCY3 and ADCY9.

Results:: Intracellular cAMP levels in BSF groups were similar in AA and CA astrocytes (AA / BSF=67+29 pmol/mg protein, and CA / BSF= 53+ 22pmol/mg protein). Stimulation with IBMX induced the expected increase in cAMP levels (AA / IBMX group=107+42pmol/mg protein; CA / IBMX= 91+32pmol/mg protein). After exposure to elevated HP for 15’ and 30’ in the presence of IBMX ,the intracellular cAMP levels increased 2-fold in AA (P< 0.01). In contrast to AA, cAMP levels of CA were not significantly affected by HP. Levels of mRNA for ADCY3 and ADCY9 were upregulated in AA astrocytes compared to CA by 1.62 and 1.47 fold respectively ( P<0.05). Exposure to elevated HP for up to 48 hours significantly increased mRNA levels of ADCY3 and ADCY9 in AA, while there was no change in CA.

Conclusions:: Our study demonstrates that endogenous intracellular cAMP levels were significantly increased in AA astrocytes compared to CA after exposure to elevated HP, a mechanical stress. Increased cAMP levels in AA is consistent with increased expression of ADCY3 and ADCY9 in AA astrocytes. Activation of the cAMP-dependent signaling pathway by pressure may contribute to increased susceptibility to elevated IOP-related stress in ONH astrocytes in AA, a population at high risk for glaucoma.

Keywords: astrocytes: optic nerve head • signal transduction • gene/expression