Abstract
Purpose: :
Regulation of intracellular cyclic AMP is one of the most ubiquitous mechanisms for regulating cellular functions. Our recent microarray and qRT-PCR analyses demonstrated upregulation of adenylyl cyclases, ADCY3 and ADCY9, in primary cultures of ONH astrocytes from African American (AA) donors compared to Caucasian Americans(CA) donors. Here we determined the effects of hydrostatic pressure (HP) on cAMP levels in astrocytes from normal (AA and CA) and glaucoma (AAG and CAG) age-matched donors.
Methods: :
Primary ONH astrocytes cultured from 6 normal AA and 6 normal CA, 4 AAG and 4 CAG 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. Two way ANOVA analysis was performed. Western blot analyses were used to determine phosphorylated CREB (pCREB) levels for astrocytes from CA and AA donors under HP. To test whether CaMK and PKA mediated phosphorylation of CREB we used specific inhibitors in mouse astrocytes exposed to HP.
Results: :
Stimulation with IBMX induced the expected increase in cAMP levels in astrocytes. After exposure to elevated HP for 15’ and 30’ in the presence of IBMX, the intracellular cAMP levels increased in AA and AAG astrocytes (P< 0.05). In contrast, cAMP levels of CA and CAG were not affected by HP. Western blot showed that pCREB increased to maximal level after 3h exposure to HP in human ONH astrocytes from AA and CA donors. Neither CaMK inhibitor KN-93 nor PKA inhibitor KT 5720 blocked phosphorylation of CREB in mouse astrocytes after 3h of exposure to HP.
Conclusions: :
Our study demonstrates that intracellular cAMP levels were significantly increased in AA and AAG astrocytes compared to CA or CAG after exposure to elevated HP, a mechanical stress. The increased cAMP levels in astrocytes induced phosphorylation of CREB after exposure to HP. The activation of cAMP signaling pathway in response to HP was PKA and CaMK independent thus the mechanism inducing CREB phosphorylation under HP is unclear. 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: astrocyte • optic nerve • signal transduction