Protein synthesis blockade differentially affects the degradation of constitutive and nicotinic receptor-induced tyrosine hydroxylase protein level in isolated bovine chromaffin cells

Elizabeth Fernández, Gale L. Craviso

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Continuous incubation of bovine adrenal chromaffin cells with the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) causes a twofold increase in the steady-state level of catalytically active tyrosine hydroxylase (TH) protein by 3-4 days. The present study examined the processes that control the time course of enzyme induction. In cells exposed to DMPP for 36 or 54 h, incorporation of [3H]leucine into TH was increased 1.9- and 2.2-fold, respectively, compared with control (non-DMPP-treated) cells. The increase correlated with a twofold rise in TH mRNA level, indicating the absence of translational control of TH synthesis by DMPP. Also absent was an effect by DMPP on the rate of degradation of TH protein because pulse-chase analysis estimated half-life for TH of 26 ± 5 h in DMPP-treated cells, a value that was (a) essentially the same as that estimated in control cells (29 ± 3 h), (b) within the same range as that estimated by approach to steady state (t(1/2) = 19 ± 4 h), which measured the decline of TH protein content from the DMPP-induced steady-state level back to the basal value during deinduction with the nicotinic antagonist hexamethonium, and (c) consistent with the time course of accumulation of TH protein to a new steady-state level in response to DMPP. However, different rates of degradation for TH protein were observed in control and DMPP-treated cells under conditions in which protein synthesis was blocked. In control cells incubated with 100 μM puromycin or 20 μM cycloheximide for 3 days, the level of catalytically active TH protein failed to decline and exhibited a half-life of ≥ 250 h. This finding indicated that TH protein was stabilized. TH protein level also failed to decline when cells were incubated for 3 days with a concentration of the transcription inhibitor α-amanitin that caused a >90% loss of TH mRNA. Thus, degradation of constitutively expressed TH protein appears to be controlled by processes dependent on ongoing transcription and translation. In contrast, the increased amount of TH induced by DMPP was not stabilized but instead underwent a decline to the basal level following addition of puromycin or cycloheximide. It is important to note, however, that the decline occurred at a slower rate (t(1/2) ≥ 45 h) than that measured during deinduction. Taken together, these data suggest that alterations in the rate of degradation of TH protein may play a role in controlling TH level when TH synthesis is blocked but not when TH synthesis is increased, such as during nicotinic receptor stimulation.

Original languageEnglish (US)
Pages (from-to)169-178
Number of pages10
JournalJournal of neurochemistry
Volume73
Issue number1
DOIs
StatePublished - 1999

Keywords

  • Approach to steady- state analysis
  • Cycloheximide
  • Induction
  • Protein half life
  • Pulse-chase analysis
  • Puromycin

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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