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First published online October 18, 2006
Journal of Experimental Biology 209, 4214-4223 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02519
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The interactive effects of hypoxia and nitric oxide on catecholamine secretion in rainbow trout (Oncorhynchus mykiss)

Brian McNeill and Steve F. Perry*

Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5, Canada


Figure 1
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  		Fig. 1. Phylogenetic analysis of rainbow trout (Oncorhynchus mykiss) NOS protein. The phylogenetic tree was constructed with commercial software (DNAMAN version 5.2.9; Lynnon Biosoft) using maximum likelihood as the distance method. Support values for nodes (100% in all cases and therefore not shown) were determined through bootstrapping of 100 pseudo-datasets. Branches are drawn to scale as the scale represents replacement of 5% of the amino acids in the protein alignment. Sequences for eNOS were obtained from rabbit (Oryctolagus cuniculus; accession AAO47084), human (Homo sapiens; accession NP_00059), dog (Canis familiaris; NP_001003158), rat (Rattus norvegicus; NP_068610) and mouse (Mus musculus; P70313). Sequences for nNOS were obtained from pufferfish (Takifugu rubripes; accession AAL82736), Japanese medaka (Oryzias latipes; accession BAD11808), zebrafish (Danio rerio; accession AAO53340), rabbit (accession AAB68663), rat (NP_434686), mouse (NP_032738), human (NP_000611) and dog (XP_534695). Sequences for iNOS were obtained from zebrafish (XP_692103), goldfish (Carassius auratus), rainbow trout (CAC8306), rat (CAB46089), mouse (AAH62378) and dog (NP_001003186).

 

Figure 2
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  		Fig. 2. Mean tissue distributions of nNOS RNA in rainbow trout (Oncorhynchus mykiss) as determined by real-time RT-PCR. The results are presented as the expression of nNOS relative to ß-actin and standardized to nNOS expression in the brain (N=5).

 

Figure 3
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  		Fig. 3. Representative fluorescence photomicrographs of rainbow trout PCV cross-sections demonstrating (A) the presence of nNOS immuno-positive cells (red) in chromaffin cells [green; areas of colocalization (arrows) appear as orange/yellow] or in close proximity to the chromaffin cells as identified by aldehyde-induced fluorescence. Cell nuclei are indicated by blue fluorescence (DAPI staining). (B) A representative western blot of trout PCV tissue demonstrating the presence of a single immunoreactive band at approximately 150 kDa (lane 1) that was absent after preabsorption of the nNOS antibody with blocking peptide (lane 2). There was no detectable red fluorescence in sections where the primary antibody was omitted (C).

 

Figure 4
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  		Fig. 4. Representative fluorescence photomicrographs of rainbow trout PCV cross-sections demonstrating (A) the presence of eNOS immuno-positive cells (red) in close proximity to the chromaffin cells (green) as identified by aldehyde-induced fluorescence. Cell nuclei are indicated by blue fluorescence (DAPI staining). (B) A representative western blot of trout PCV tissue demonstrating the presence of a single immunoreactive band at approximately 140 kDa (lane 1) that was absent after preabsorption of the eNOS antibody with blocking peptide (lane 2). There was no detectable red or green fluorescence in sections not treated with aldehyde and on which the primary antibody was omitted (C).

 

Figure 5
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  		Fig. 5. The effects of 30-min exposure to acute hypoxia (PO2<5.99 kPa) on plasma (A,B) total catecholamine levels (filled bars; N=6) and (C,D) NOX concentrations (filled bars; N=6). Unfilled bars are basal levels prior to hypoxia exposure. In all cases, fish were exposed twice-daily to acute hypoxia for a total of 4 days, but in one series (C,D) paired blood samples were withdrawn only once each on day 1 and 4. Values are shown as means ± 1 s.e.m. A dagger denotes a significant difference (P<0.5) between the pre-hypoxia values (unfilled bars) and hypoxia exposure (filled bars). An asterisk denotes a significant difference (P<0.5) in plasma catecholamine levels during hypoxia from value of the peak response (day 2, PM). A double dagger denotes a significant difference (P<0.5) in basal or hypoxia-evoked NO levels from the lowest values (Day 1, PM).

 

Figure 6
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  		Fig. 6. The effects of in situ electrical stimulation on (A) total catecholamine secretion (sum of noradrenaline plus adrenaline) and (B) perfusate NOX levels in preparations derived from fish repeatedly exposed to hypoxia (unfilled bars; N=9) or from control fish (filled bars; N=9). Following the stabilization period, the preparations were perfused with control saline or saline containing a cocktail of NOS inhibitors (cross-hatched bars) and electrically stimulated at 30 V and 8 Hz. Values are shown as means ± 1 s.e.m. A dagger denotes a significant difference (P<0.5) between pre-experimental (Pre) and stimulated samples. An asterisk denotes a significant difference (P<0.5) between the hypoxia-treated preparations with or without NOS inhibitors.

 

Figure 7
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  		Fig. 7. The relative expression of (A) nNOS (N=10) and (B) eNOS (N=7) protein in the PCV of rainbow trout (Oncorhynchus mykiss) that were either subjected to 4 days of repeated hypoxia or continued normoxia. Values are shown as means ± s.e.m. An asterisk denotes a significant difference (P<0.5) between the control and hypoxia-treated groups.

 

Figure 8
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  		Fig. 8. The effects of repeated hypoxia exposures on the relative expression of nNOS mRNA in various tissues as determined by realtime RT-PCR. Values are shown as means ± 1 s.e.m. (N=5). In each tissue, an asterisk denotes a significant difference (P<0.5) in the relative levels of mRNA between control and hypoxia-exposed fish.

 

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© The Company of Biologists Ltd 2006