Pattern-specific sustained activation of tyrosine hydroxylase by intermittent hypoxia: role of reactive oxygen species-dependent downregulation of protein phosphatase 2A and upregulation of protein kinases

Abstract

We investigated the role of protein phosphatases (PP) and protein kinases in tyrosine hydroxylase (TH) activation by two patterns of intermittent hypoxia (IH) in rat brainstem. Rats exposed to either IH(15s) (15 s, 5% O(2); 5 min, 21%O(2)) or IH(90s) (90 s each of 10% O(2) & 21%O(2)) for 10 days were used. IH(15s) but not IH(90s) caused a robust increase in TH activity, dopamine (DA) level, and TH phosphorylation at Ser-31 and Ser-40 in the medulla but not in the pons. Likewise, IH(15s) but not IH(90s) decreased activity and expression of protein phosphatase 2A (PP2A) and increased activity of multiple protein kinases. In vitro dephosphorylation with PP2A nearly abolished IH(15s)-induced increase in TH activity. IH(15s) increased generation of reactive oxygen species (ROS) in brainstem medullary regions which was nearly threefold higher than that evoked by IH(90s). Antioxidants prevented IH(15s)-induced downregulation of PP2A and increases in multiple protein kinase activity with subsequent reversal of serine phosphorylation of TH, TH activity, and DA to control levels. These findings demonstrate that IH in a pattern-specific manner activates TH involving ROS-mediated sustained increase in TH phosphorylation via downregulation of PP2A and upregulation of protein kinases.

FIG. 1.

Effects of two patterns of intermittent hypoxia (IH) on tyrosine hydroxylase (TH) activity and dopamine (DA) levels in the rat brainstem. Changes in TH activity as a function of reaction time are shown (A). TH activity was measured in the whole brainstem extracts of rats exposed to either IH_15s (alternating cycles of 15 s of 5% O₂ and 5 min of 21% O₂) or IH_90s (alternating cycles of 90 s of 10% O₂ and 90 s of 21% O₂) or normoxia (NOR) for 10 days, as described under “Methods”. Effects of IH_15s and IH_90s on TH activity (B) and on DA levels (C) in the whole brainstem are shown. For DA analysis, brainstems were extracted with 0.1 N HCLO₄. DOPA formed during TH reaction and DA level in the whole brainstem were determined by HPLC combined with electrochemical detection as described in “Methods”. TH activity is expressed as nanomoles of DOPA formed per min per mg protein, and DA as nanomoles per gram tissue. A comparison of the effect of IH_15s on TH activity in the dorsal and ventral medullary (D; left panel) and pontine (E; right panel) brainstem regions is shown. All measurements were made in triplicate and average data are shown as mean ± S.E. (n = 7 rats in each group). Asterisks denote p < 0.01. n.s., not significant (p > 0.05).

FIG. 2.

Effects of IH_15s on total and serine phosphorylated forms of TH protein expression in the dorsal and ventral medullary brainstem regions. Equal amounts of proteins (20 μg) from normoxic and IH_15s extracts were used for immunoblot analysis. Upper panels: Representative examples of immunoblots of total and serine phosphorylated forms of TH (P-TH). Bottom panels: IH-induced fold change in total or serine phosphorylated forms of TH relative to normoxic control. Other experimental details are as described in “Methods”. All measurements were made in duplicate and average data are shown as mean ± S.E. (n = 7 rats in each group). Asterisks denote p < 0.01; n.s., not significant (p > 0.05).

FIG. 3.

Changes in PP2A enzyme activity and protein phosphatase (PP) expression in the dorsal and ventral medulla by IH_15s. Effect of IH on PP2A enzyme activity is shown (A). PP2A activity was determined by procedures as described under “Methods” and is expressed as micromoles per minute per mg protein. Representative immunoblots showing IH_15s-induced changes in PP expression in the dorsal (B) and ventral (C) medulla are presented. Equal amounts of proteins (20 μg) from normoxic (NOR) and IH_15s medullary extracts were used for immunoblot analysis. Changes in PP expression as percent of normoxia are shown in (D). All measurements were made in triplicate and average data are shown as mean ± S.E. (n = 7 rats in each group). Asterisks denote p < 0.01.

FIG. 4.

Reversal of IH_15s-induced increase in TH activity by PP2A. TH activity was determined in the extracts of dorsal and ventral medulla derived from rats exposed to normoxia (NOR) or IH_15s for 10 days before and after dephosphorylation. For dephosphorylation, aliquots of the medullary extracts were treated with 0.2 units of the catalytic subunit of PP2A for 15 min, as described in the “Methods”. All measurements were made in triplicate and average data are shown as mean ± S.E. (n = 7 rats in each group). Asterisks denote p < 0.01.

FIG. 5.

Activation of protein kinases by IH_15s. Effects of IH_15s on the activity of protein kinase A (PKA; A), Ca²⁺/calmodulin-dependent kinase II (CaMKII; B), and MAP kinase/extracellular signal regulated kinase 1 and 2 (ERK1/2; C) in the dorsal and ventral medulla are shown. Active PKA and CaMKII were determined using commercially available Kinase Assay Kit as per manufacturer's instructions. Activity of ERK1/2 was assessed by immunoblot analysis using antibodies specific for total- and phospho-ERK1/2 (P-ERK1/2). All measurements were made in triplicate and average data are shown as mean ± S.E. (n = 7 rats in each group). Asterisks denote p > 0.01.

FIG. 6.

IH-evoked ROS generation and effects of antioxidants on IH-induced ROS generation, increase in TH activity, and elevation in DA level in the dorsal and ventral medulla. The effects of IH_15s (A) and IH_90s (B) on T-BARS level in dorsal and ventral medullary homogenates are shown. MnTMPyP, a membrane permeable superoxide anion mimetic and N-acetyl-L-cysteine (NAC; a scavenger of reactive oxygen species) were administered (i.p.) daily prior to exposure of rats to either normoxia (NOR) or IH as described in “Methods”. T-BARS level was expressed as nanomoles of malondialdehyde (MDA) formed per mg protein. Effects of MnTMPyP and NAC on IH_15s-induced increase in TH activity (C) and elevation of DA (D) are shown. DA was measured in whole brainstem extracts. All measurements were made in triplicate and average data are shown as mean ± S.E. (n = 7 rats in each group). Asterisks denote p < 0.01.

FIG. 7.

Reversal of IH_15s-induced decrease in PP2A activity and the increase in PKA and CaMKII activity and serine phosphorylation of TH by antioxidants. The effects of MnTMPyP on IH_15s-evoked decrease in PP2A activity (A) and increases in PKA (B), CaMKII (C), and phosphorylation of TH at S-19, S-31 and S-40 (P-TH; D) in the dorsal and ventral medullary brainstem regions are presented. Other experimental details are as described in the legends of Figs. 3 and 5. The data are expressed as percent of normoxic control. All measurements were made in triplicate and average data are shown as mean ± S.E. (n = 7 rats in each group). Asterisks denote p < 0.01.

FIG. 8.

Schematic presentation of ROS-dependent downregulation of PP2A and upregulation of protein kinases in IH_15s-induced activation of TH.