Tyrosine Hydroxylase Phosphorylation in Catecholaminergic Brain Regions: A Marker of Activation following Acute Hypotension and Glucoprivation

Abstract

The expression of c-Fos defines brain regions activated by the stressors hypotension and glucoprivation however, whether this identifies all brain sites involved is unknown. Furthermore, the neurochemicals that delineate these regions, or are utilized in them when responding to these stressors remain undefined. Conscious rats were subjected to hypotension, glucoprivation or vehicle for 30, 60 or 120 min and changes in the phosphorylation of serine residues 19, 31 and 40 in the biosynthetic enzyme, tyrosine hydroxylase (TH), the activity of TH and/or, the expression of c-Fos were determined, in up to ten brain regions simultaneously that contain catecholaminergic cell bodies and/or terminals: A1, A2, caudal C1, rostral C1, A6, A8/9, A10, nucleus accumbens, dorsal striatum and medial prefrontal cortex. Glucoprivation evoked phosphorylation changes in A1, caudal C1, rostral C1 and nucleus accumbens whereas hypotension evoked changes A1, caudal C1, rostral C1, A6, A8/9, A10 and medial prefrontal cortex 30 min post stimulus whereas few changes were evident at 60 min. Although increases in pSer19, indicative of depolarization, were seen in sites where c-Fos was evoked, phosphorylation changes were a sensitive measure of activation in A8/9 and A10 regions that did not express c-Fos and in the prefrontal cortex that contains only catecholaminergic terminals. Specific patterns of serine residue phosphorylation were detected, dependent upon the stimulus and brain region, suggesting activation of distinct signaling cascades. Hypotension evoked a reduction in phosphorylation in A1 suggestive of reduced kinase activity. TH activity was increased, indicating synthesis of TH, in regions where pSer31 alone was increased (prefrontal cortex) or in conjunction with pSer40 (caudal C1). Thus, changes in phosphorylation of serine residues in TH provide a highly sensitive measure of activity, cellular signaling and catecholamine utilization in catecholaminergic brain regions, in the short term, in response to hypotension and glucoprivation.

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function

“…Blood samples collected 120 min post HDZ were centrifuged for 10 min at 1800 rpm (4 1C) and then 10 min at 2700 rpm (4 1C) to separate plasma from red blood cells, leukocytes, and platelets and stored at À80 1C. Plasma catecholamines were extracted and measured on a HTEC-500 Complete Stand-Alone HPLC-ECD system (Eicom Corporation, Kyoto Japan) as described previously (Bobrovskaya et al, 2010;Damanhuri et al, 2012;Parker et al, 2013) with slight modifications on the method of Anton and Sayre (1962). The specific retention times were determined using adrenaline and noradrenaline standards.…”

“…We also assessed the activation of adrenal chromaffin cells using c-Fos-ir and sensitive measures of catecholamine synthesis, i.e. phosphorylation of tyrosine hydroxylase which indicate increased capacity for catecholamine release (Damanhuri et al, 2012) across multiple time points.…”

Early life peripheral lipopolysaccharide challenge reprograms catecholaminergic neurons

“…In short-term in vivo stress models (less than 1 hour) there were increases in Ser31 and Ser40 phosphorylation in response to social defeat and footshock2329 and increases in Ser31 phosphorylation alone in response to restraint, hypotension and glucoprivation2230. In contrast to short-term stressor results, the effect of the neonatal LPS challenge on the LC produced much more robust responses in relation to Ser40 phosphorylation and produced a dramatic change in Ser19 phosphorylation that was not seen in the other models.…”

“…The increased level of TH protein was accompanied by increased phosphorylation of Ser31 and Ser40, two sites that have been shown to be directly associated with TH activation in vivo 7112223242526. This would explain the very significant increase in the LC TH activity.…”

“…The SN, VTA and LC were dissected from the coronal sections as previously described which will separate out the different brain sections such that they only have the ascribed catecholaminergic nuclei2223. The SN, VTA and LC samples were separately processed as previously described23.…”