Retinal melatonin production: role of proteasomal proteolysis in circadian and photic control of arylalkylamine N-acetyltransferase.
OBJECTIVE
Dynamic day-night changes in melatonin synthesis are regulated by changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AA-NAT]). Similarly, a light-induced decrease in AA-NAT activity at night rapidly suppresses melatonin synthesis. The purpose of the current study was to test the hypothesis that in vivo changes of AA-NAT activity in chicken retina homogenates parallel changes in AA-NAT protein. This led to examination of the role of proteasomal proteolysis in the regulation of retinal AA-NAT activity and protein levels.


METHODS
Chickens, entrained to a 12-hour light-12-hour dark cycle, were assessed under various lighting conditions, in some cases after in vivo intravitreal administration of the protein synthesis inhibitor cycloheximide or lactacystin, an inhibitor of the 20S proteasome. Tissue homogenates were prepared, AA-NAT enzyme activity was measured, and immunoreactive protein was estimated by Western blot using an anti-chicken AA-NAT(1-21) serum.


RESULTS
The abundance of AA-NAT protein in both the retina and pineal gland exhibited a daily rhythm that was statistically indistinguishable from that of AA-NAT's activity measured in tissue homogenates. Acute exposure to light at night rapidly decreased AA-NAT protein and activity in a parallel fashion. Administration of cycloheximide at night decreased retinal AA-NAT activity in darkness and enhanced the effect of light. The light-evoked suppression of retinal AA-NAT protein and activity was blocked by intravitreal injection of lactacystin, which also was found to increase AA-NAT activity, either at night or during the daytime.


CONCLUSIONS
AA-NAT activity measured in tissue homogenates reflects the steady state level of enzyme protein. AA-NAT protein in the retina turns over rapidly, reflecting a balance of de novo synthesis and proteasomal proteolysis. The suppressive effects of light at night are due primarily to enhanced AA-NAT proteolysis.
Iuvone
P Michael
PM
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322-3090, USA. miuvone@pharm.emory.edu
Brown
Audra D
AD
Haque
Rashidul
R
Weller
Joan
J
Zawilska
Jolanta B
JB
Chaurasia
Shyam S
SS
Ma
Minhui
M
Klein
David C
DC
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism*; D015153:Blotting, Western; D002645:Chickens; D002940:Circadian Rhythm / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D008297:Male; D008550:Melatonin / Q000096:biosynthesis*; D009097:Multienzyme Complexes / Q000502:physiology*; D010775:Photic Stimulation; D010870:Pineal Gland / Q000201:enzymology; D046988:Proteasome Endopeptidase Complex; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits; D012160:Retina / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects
D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D011500:Protein Synthesis Inhibitors; C067713:lactacystin; D003513:Cycloheximide; D001191:Arylamine N-Acetyltransferase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008550:Melatonin; D000111:Acetylcysteine