ZT h

Figure 1. Daily rhythms of TPH and AA-NAT activities in chick retina. Retinas were collected at the Zeitgeber times (ZT) indicated during the 12h light—12h dark cycle (LD, lights on at ZT 0). TPH activity was estimated from the in situ accumulation of 5-hydroxytryptophan (5HTP) 30 min following inhibition of aromatic L-amino acid decarboxylase activity with m-hydroxybenzylhydrazine (mHBH)(38). AA-NAT activity was measured in retinal homogenates using tryptamine and acetyl coenzyme A as substrates (40). Adapted from Thomas and Iuvone (38) and Iuvone and Alonso-Gomez (23).

Figure 2. Effect of constant light on the rhythms of TPH and AA-NAT activities. Retinas were dissected 30min following injection of rnHBH at midday (ZT 6) and midnight (ZT 18). On day 1, animals were exposed to the entrained LD cycle. Constant light (LL) began on the second day. *p < 0.01 vs ZT6. Adapted from Thomas and Iuvone (38).

Figure 2. Effect of constant light on the rhythms of TPH and AA-NAT activities. Retinas were dissected 30min following injection of rnHBH at midday (ZT 6) and midnight (ZT 18). On day 1, animals were exposed to the entrained LD cycle. Constant light (LL) began on the second day. *p < 0.01 vs ZT6. Adapted from Thomas and Iuvone (38).

Zeitgeber Time (hr) 18 24 6 12 18 24 6 12 1fl 24

Zeitgeber Time (hr) 18 24 6 12 18 24 6 12 1fl 24

Rna And Melatonin

Figure 3. Rhythms of AA-NAT mRNA and activity in LL. Chickens were housed in LD and were then transferred to LL. The open horizontal bar indicates when lights were on and the vertical lines indicate the subjective day/night transitions. Retinas were taken at the indicated times and processed for RNA analysis. The top panels show representative Northern blot analyses of AA-NAT and actin mRNAs. Each lane contains 20^g of total RNA. The bottom panels represent the quantitative analysis of the Northern blots (•-•) and the levels of AA-NAT activity (o-o) in each experimental group. The abundance of the AA-NAT transcript has been normalized to actin mRNA, to correct for variations in loading. All values are expressed relative to the first ZT 6 time point values. AA-NAT activity at ZT 6 was 26 ± 3pmoles/min/mg protein. Adapted from Bernard et al. (2).

18 24 6 12 18 24 6 12 18 24 Zeitgeber Time (hr)

Figure 3. Rhythms of AA-NAT mRNA and activity in LL. Chickens were housed in LD and were then transferred to LL. The open horizontal bar indicates when lights were on and the vertical lines indicate the subjective day/night transitions. Retinas were taken at the indicated times and processed for RNA analysis. The top panels show representative Northern blot analyses of AA-NAT and actin mRNAs. Each lane contains 20^g of total RNA. The bottom panels represent the quantitative analysis of the Northern blots (•-•) and the levels of AA-NAT activity (o-o) in each experimental group. The abundance of the AA-NAT transcript has been normalized to actin mRNA, to correct for variations in loading. All values are expressed relative to the first ZT 6 time point values. AA-NAT activity at ZT 6 was 26 ± 3pmoles/min/mg protein. Adapted from Bernard et al. (2).

3.3. Effects of Acute Light Exposure on TPH and AA-NAT mRNAs and

Enzyme Activities

Light is known to suppress melatonin production by suppressing AANAT activity in both the pineal gland and retina (20,24). In the chicken, it has been found that light exposure during the middle of the dark phase of the LD cycle reduces retinal melatonin levels to daytime values within 1 hour (19). This treatment elicits only small decreases (~30%) of TPH and AA-NAT mRNAs (2,12). Acute light exposure also has little inhibitory effect (~30%) on TPH activity (Figure 4). In contrast, AA-NAT activity is dramatically reduced (~80%) by acute light exposure (Figures 4 and 5). Thus, the effects of acute light exposure on TPH activity appear to reflect mainly changes of mRNA level, while that on AA-NAT activity is elicited by

Melatonin Receptor Agonist

Figure 4. Differential effects of acute light exposure on nocturnal TPH and AA-NAT activities. Animals were exposed to fluorescent room light (2 x 10-4W/cm2) during middark phase (ZT 18) for 60min (Dark-» Light). After the initial 30min of light exposure, they were injected with mHBH. Retinas were dissected in light 30min after injection. Control animals (dark) were treated identically except that they were not exposed to light. Retinas were also obtained from mHBH-treated animals during midlight phase (ZT 6). Adapted from Thomas and Iuvone (38).

Figure 4. Differential effects of acute light exposure on nocturnal TPH and AA-NAT activities. Animals were exposed to fluorescent room light (2 x 10-4W/cm2) during middark phase (ZT 18) for 60min (Dark-» Light). After the initial 30min of light exposure, they were injected with mHBH. Retinas were dissected in light 30min after injection. Control animals (dark) were treated identically except that they were not exposed to light. Retinas were also obtained from mHBH-treated animals during midlight phase (ZT 6). Adapted from Thomas and Iuvone (38).

both decreased mRNA levels and post-transcriptional regulation of enzyme activity. The post-transcriptional regulatory mechanism may involve decreased cyclic AMP levels in the photoreceptor cell resulting in a destabilization of AA-NAT (1). By analogy to the rat pineal gland (16), the destabilization of AA-NAT may result in its proteolysis.

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