Signal transduction pathways and tyrosine hydroxylase regulation in the adrenal medulla following glucoprivation: An in vivo analysis

by Larisa Bobrovskaya, Hanafi A. Damanhuri, Lin Kooi Ong, Jennifer J. Schneider, Phillip W. Dickso, Peter R. Dunkley, and  Ann K. Goodchild

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Catecholamine synthesis in the adrenal medulla is dependent on the rate-limiting enzyme, Tyrosine Hydroxylase (TH). When chromaffin cells of the adrenal medulla release catecholamines such as epinephrine (Ad) and norepinephrine (NAd), an increase of catecholamine synthesis follows in order to keep intracellular catecholamine levels steady. An increase of catecholamine synthesis to maintain homestatic conditions suggests further regulation of the synthesis and activity of TH. TH activity is regulated by phosphorylation of residue Ser40 after feedback inhibition by catecholamines. Furthermore, Phos-Ser31 residue can double TH activity alone, while phosphorylated Ser31 and phosphorylated Ser19 is known to increase rate of Ser40 phosphorylation, which in turn increases TH activity.

The mechanisms behind TH regulation had only been experimented in vitro or in situ prior to this study.  Therefore, this study sought to identify various signaling pathways that regulate TH activity in conscious rats following a physiological stimuli which increases plasma levels of catecholamines. In this case, the physiological stimuli was a single episode of 2-deoxy-d-glucose (2DG), used to evoke glucoprivation to promote increases in plasma Ad and thereon increasing medullary TH levels. By increasing plasma catecholamine levels, the neurotransmitters or signaling systems involved in increasing medullary TH levels during glucoprivation could be analyzed. 


Materials and Methods: One day prior to the experiment, Sprague-Dawley rats (n=44 total) were housed in a temperature-controlled room with free access to food and water.  On the day of the study, rats were injected intraperitoneally  with either 2DG or saline as a control. Food and water were immediately removed from the cage following injection, and the rats were sacrificed after either 5, 20, 60 mins, or 24 hours. 

• Blood samples were taken for catecholamine analysis using a liquid-liquid extraction assay.
• In some cases, adrenal medullas were separated from cortices, and medullary membranes were immunoblotted with antibodies for analysis of phosphorylated Protein Kinase A (PKA), Protein Kinase C (PKC), MAPK (Mitogen-activated Protein Kinase), and MAPK/Cyclin-dependent kinase (CDK) substrates
• Whole adrenals were processed and run on SDS-PAGE to measure TH phosphorylation of Ser 19, 31, and 40, expressed as ratios to total TH. 

Results:

1. Catecholamine and BGL: The results revealed significant increases in Ad and Nad in the plasma 20m after 2DG injection, but not 24h after injection. There were also significant increases in blood glucose levels after 20 and 60m of 2DG injection 
2. Protein Kinase Activation: Phosphorylated PKA levels were significantly elevated  after 20 and 60m of 2DG injection, however no significant changes in PKC levels were observed. There was no significant difference of MAPK or MAPK/CDK phosphorylation at 20m, but there was  a significant increase at 60m.  
3. TH residues to total TH protein: Ser19 phosphorylation was not significantly changed at 5, 20, or 60m. Ser40 appeared to be activated after 20m and then declined with time, returning to baseline at 24h. Ser31 increased at 20m, reached maximum residue/protein at 60m, and returned to baseline at 24h. Total TH was only significantly increased 24h after 2DG injection.

Discussion: 

The data of this experiment suggested glucoprivation evokes increases in Ad and NAd plasma levels, which resulted in increase plasma glucose levels. Furthermore, activation of signalling pathways varied with respect to time, in which Ser40 was activated and declined sooner than Ser31 was, and changes in activation of Ser19 to total protein remained insignificant. Activation of PKA and MAPK/CDK substrate phosphorylation after 20 and 60m of 2DG injection but not PKC indicates effects of glucoprivation on specific protein kinases involved in TH activity. The significant increases in total TH protein after 24h of glucoprivation suggests a response that restores and also increases Ad in the adrenal medulla. 

This study is relevant to my project due to the importance of catecholamines in the cardiovascular response to exercise, which is a metabolic stressor. This study provided insight to the regulatory mechanisms that maintain TH and epinephrine levels within the adrenal medulla following a metabolic stressor involved in the production and release of catecholamines. Since the pathway of catecholamine synthesis is dependent on TH, activation of different protein kinases involved in TH phosphorylation (such as PKA or MAPK) may also have a notable role in the production and replenishment of catecholamines. Without the effects of kinases on TH phosphorylation, we would expect to see altered physiological responses and, more specific to our studies, altered catecholamine synthesis and secretion which in turn would change cardiovascular responses such as heart rate, contraction, conduction, and vasodilation during periods of physical activity. 

-NSS