GRK2 Up-Regulation Creates a Positive Feedback Loop for Catecholamine Production in Chromaffin Cells

Abstract

Elevated sympathetic nervous system (SNS) activity aggravates several diseases, including heart failure. The molecular cause(s) underlying this SNS hyperactivity are not known. We have previously uncovered a neurohormonal mechanism, operating in adrenomedullary chromaffin cells, by which circulating catecholamine (CA) levels increase in heart failure: severe dysfunction of the adrenal α2-adrenergic receptors (ARs) due to the up-regulation of G protein-coupled receptor-kinase (GRK)-2, the kinase that desensitizes them. Herein we looked at the potential signaling mechanisms that bring about this GRK2 elevation in chromaffin cells. We found that chronic CA treatment of either PC12 or rat primary chromaffin cells can in itself result in GRK2 transcriptional up-regulation through α2ARs-Gi/o proteins-Src-ERK1/2. The resultant GRK2 increase severely enhances the α2AR desensitization/down-regulation elevating not only CA release but also CA biosynthesis, as evidenced by tyrosine hydroxylase up-regulation. Finally, GRK2 knockdown leads to enhanced apoptosis of PC12 cells, indicating an essential role for GRK2 in chromaffin cell homeostasis/survival. In conclusion, chromaffin cell GRK2 mediates a positive feedback loop that feeds into CA secretion, thereby enabling the adrenomedullary component of the SNS to turn itself on.

Figure 1.. Chronic NE treatment of PC12 cells up-regulates GRK2 but not GRK5.

α₂AR-transfected PC12 cells were treated with 10 μM NE or vehicle (control) either once for 6 hours or every 6 hours (repeatedly) for a total period of 24 hours (NE 24 h) in the presence or absence of 10 μM RX821002 (RX + NE). Nontransfected NE-treated cells were also included in the analysis (NE Nontransf., NT). At the end of the indicated treatment periods, cells were harvested and total RNA was isolated to perform real-time PCR for GRK2 mRNA quantitation (A), or protein extracts were prepared to perform Western blotting for GRK2 protein quantitation (B). Blots for GAPDH serving as the loading control are also shown. C, The densitometric quantitation of GRK2 protein induction. *, P < .05 vs control-6 hours (n = 5 independent experiments/condition). D, GRK5 mRNA levels as measured by real-time PCR in α₂AR-transfected PC12 cells treated with 10 μM NE or vehicle (control) either once for 6 hours or every 6 hours (repeatedly) for a total period of 24 hours (NE 24 hours). No significant differences were observed among the treatments (n = 5 independent determinations/condition).

Figure 2.. Chronic NE-induced GRK2 up-regulation results in enhanced α₂AR desensitization and down-regulation in primary chromaffin cells.

A, In vitro NE (top panel) and Epi (bottom panel) secretion from cultured rat primary chromaffin cells treated with 10 μM NE (NE) or vehicle (control) for 24 hours (repeated application every 6 hours), prior to being placed in the secretion assay buffer. The cells were left to settle in the assay buffer for 30 minutes and then were treated with 10 μM UK14304 for another 30 minutes. At the end of this period, the buffer was changed to wash out UK14304, and then 50 μM nicotine was applied in a fresh buffer, either alone (Nicotine) or together with another 10 μM UK14304 challenge (U-K14304 + Nicotine). No differences were observed between non-UK14304-pretreated cells (data not shown). *, P < .05 vs control/nicotine or NE/UK14304 + nicotine (n = 5 independent experiments). B, Saturation ligand-binding studies using [³H]RX821002 on plasma membrane preparations from these cells. Membrane preparations from cells immediately after isolation (upon plating) were also analyzed for comparison (control 0 h). Nonspecific binding was determined in the presence of 0.4 mM phentolamine. *, P < .05 vs either of the other groups (n = 5 independent experiments/group). Data are expressed as mean ± SEM. C, Western blotting for GRK2 in whole-protein extracts from these cells. Representative blots from three independent experiments are shown, including GAPDH as loading control.

Figure 3.. Chronic NE-induced GRK2 up-regulation is mediated transcriptionally by G_i/o proteins and ERKs.

α₂AR-transfected PC12 cells were treated with 10 μM NE or vehicle (control) for 6 hours in the presence or absence of 5 μg/mL actinomycin-D (Actino + NE), 100 ng/mL pertussis toxin (PTX + NE), 1 μM Src-inhibtor-1 (SI-1 + NE), or 10 μM PD98059 (PD + NE). At the end of the indicated treatment periods, cells were harvested and total RNA was isolated to perform real-time PCR for GRK2 mRNA quantitation (A) or protein extracts were prepared to perform Western blotting for GRK2 protein quantitation (B). B, Representative Western blots for GRK2, including GAPDH as loading control. C, The densitometric quantitation of GRK2 protein induction. *, P < .05 vs all other groups (n = 5 independent experiments/condition).

Figure 4.. GRK2 promotes chronic NE-induced CA biosynthesis and survival in chromaffin cells.

A, α₂AR-expressing PC12 cells were transfected with GRK2- or scrambled (Scr)-siRNA and simultaneously treated with 10 μM NE or vehicle (control) for 24 hours (repeatedly every 6 h). At the end of the treatment period, cells were harvested and total RNA was isolated to perform real-time PCR for TH mRNA quantitation. *, P < .05 vs control (n = 5 independent experiments/condition). B and C, Apoptotic cell death of α₂AR-expressing PC12 cells at 24 hours after the transfection with GRK2- or scrambled (Scr)-siRNA, as measured by TUNEL. Representative images of TUNEL-positive nuclei are shown in panel B, and the quantitation in panel C. *, P < .05 vs Scr, (n = 5 fields scanned per group).

Figure 5.. Schematic illustration of the chronic CA-elicited signaling pathway leading to GRK2 up-regulation in chromaffin cells.

See text for details and molecular acronym definitions.