Assessment of endogenous CRF secretion by CRF release from the rat hypothalamus in vitro.

The literature on the control mechanism of corticotrophin releasing factor (CRF) secretion is reviewed and the limitations of CRF assays are discussed. A new specific in vitro CRF assay is described which does not respond to vasopressin because the pituitary tissue is not preincubated. The technique does not require surgical or pharmacological pretreatment since endogenous CRF release due to experimentation is prevented by handling the donor rats until 'adaptation' to handling occurs (after two weeks). The results show that the assay gives reliable estimates of the CRF potency of hypothalamic extracts. Although endogenous CRF release is usually assessed by changes in hypothalamic CRF content, this is not always a reliable index of CRF release. There is also considerable confusion about the effects of various pretreatments on CRF content. It was therefore decided to test whether the initial release of CRF from isolated hypothalami in vitro was able to reflect the changes in CRF release in vivo under various conditions. The results demonstrate that in vitro CRF release during two fifteen minute incubation periods immediately after killing is able to reflect the changes in endogenous CRF secretion (assessed by plasma corticosterone levels) under all the conditions tested (i.e. acute ether stress, during the circadian cycle and after chronic betamethasone and reserpine treatments in basal and acute stress conditions) and is therefore a valuable tool for assessing the effects of various treatments on acute CRF release. These results also provide further evidence for control of pituitary/adrenocortical activity by changes in CRF secretion. The circadian rise in plasma corticosterone is preceded by a threefold rise in CRF secretion. Chronic betamethasone treatment blocks basal and stress-induced CRF release, whereas long-term reserpine treatment only causes adaptation to vehicle-injection stress. Acute CRF release was unimpaired in aged rats. A corticotrophin inhibiting factor may be released from the hypothalamus under certain conditions

Molecular and biochemical responses of hypoxia exposure in Atlantic croaker collected from hypoxic regions in the northern Gulf of Mexico

<div><p>A major impact of global climate change has been the marked increase worldwide in the incidence of coastal hypoxia (dissolved oxygen, DO<2.0 mg l^-1). However, the extent of hypoxia exposure to motile animals such as fish collected from hypoxic waters as well as their molecular and physiological responses to environmental hypoxia exposure are largely unknown. A suite of potential hypoxia exposure biomarkers was evaluated in Atlantic croaker collected from hypoxic and normoxic regions in the northern Gulf of Mexico (nGOM), and in croaker after laboratory exposure to hypoxia (DO: 1.7 mg l^-1). Expression of hypoxia-inducible factor-α, <i>hif-α</i>; neuronal nitric oxide synthase, <i>nNOS</i>; and insulin-like growth factor binding protein, <i>igfbp</i> mRNAs and protein carbonyl (PC, an oxidative stress indicator) content were elevated several-fold in brain and liver tissues of croaker collected from nGOM hypoxic sites. All of these molecular and biochemical biomarkers were also upregulated ~3-10-fold in croaker brain and liver tissues within 1–2 days of hypoxia exposure in controlled laboratory experiments. These results suggest that <i>hif-α</i>s, <i>nNOS</i> and <i>igfbp-1</i> transcripts and PC contents are useful biomarkers of environmental hypoxia exposure and some of its physiological effects, making them important components for improved assessments of long-term impacts of environmental hypoxia on fish populations.</p></div

Hypoxia-induced expression of CYP1A protein determined by Western blot analysis.

<p>Effects of 1, 2 and 4 weeks hypoxia (dissolved oxygen, DO: 1.7 mg/L) exposure on CYP1A protein expression (<b>A</b>) and relative protein levels (<b>B</b>), and actin protein levels (<b>C</b>) in croaker liver. Each value represents the mean±S.E.M (N = 8–10, tissues were randomly sampled from individual fish for measurements). Asterisk indicates significant difference from normoxic controls (Student’s <i>t</i>-test, *p<0.05, **p<0.01). Note: exposure duration only refers to period fish were exposed to target DO; fish were previously exposed to declining DO for additional 2-day adjustment period. The positions of Western blot protein standard marker (PM) are indicated on the left. No significant difference was detected in actin protein levels between the treatment groups. CTL, control; HYP, hypoxia.</p

<i>igfbp-1</i> mRNA levels in livers of Atlantic croaker collected from normoxic and hypoxic sites in the nGOM and after exposure to hypoxia in the laboratory.

<p>Croaker collected from normoxic (N1, N2) and hypoxic (F3, F4, C8, C9) sites in August 2007 (i), July 2008 (ii), and August 2012 (iii) in the nGoM (A). Asterisks denote significant differences between normoxic (reference) and hypoxic sites (nested ANOVA, **<i>p</i><0.01). The horizontal lines represent mean values, N = 7–8. Individual site differences are indicated with different letters (Fisher’s PLSD, <i>p</i><0.05). (B, C) Expression of <i>igfbp-1</i> mRNA levels and C<i>t</i> values of <i>18S</i> rRNA in livers of croaker exposed to laboratory hypoxia. Effects of 7-day laboratory exposure to normoxia (DO: ~6.5 mg l^-1, white bars), hypoxia (HYP, DO: 1.7 mg l^-1, black bars) and recovery period on <i>igfbp-1</i> mRNA levels in croaker liver. Each value represents the mean±SE (N = 7–8). Differences in the relative mRNA levels between the start of the experiment (CTL, control) and each treatment were tested by Dunnett’s test, *<i>p</i><0.05. ‘†’ indicates significant difference from normoxic controls (Student’s <i>t</i>-test, <i>p</i><0.05).</p

Atlantic croaker sampling sites and brain <i>hif-α</i> mRNA levels in croaker collected from hypoxic and normoxic sites in the nGoM.

<p>(A) Location of four hypoxic sites (C8, C9, F3 and F4) in the coastal region in the nGoM and two normoxic reference sites (N1 and N2) to the east of the Mississippi Delta where croaker were collected in August, 2007; July, 2008; and August, 2012. The map including sampling sites was generated using Ocean Data View software. Black circles indicate sampling sites. Insert bar graphs indicate dissolve oxygen (DO: mg l^-1) levels during sampling period. (B, C) Relative <i>hif-1α</i> (B) and <i>hif-2α</i> (C) mRNA levels in brains of croaker collected from normoxic (N1, N2) and hypoxic (F3, F4, C8, C9) sites in August 2007 (i), July 2008 (ii), and August 2012 (iii) in the nGoM. Asterisks denote significant differences between normoxic (reference) and hypoxic sites (nested ANOVA, **<i>p</i><0.01). The horizontal lines represent mean values, N = 8. Individual site differences are indicated with different letters (Fisher’s PLSD, <i>p</i><0.05). DO, dissolved oxygen (mg l^-1).</p

Expression of <i>nNOS</i> mRNA in the brains and plasma NOx levels in Atlantic croaker exposed to laboratory hypoxia.

<p>Effects of 7-day laboratory exposure to normoxia (dissolved oxygen, DO: ~6.5 mg/L, white bars), hypoxia (HYP, DO: 1.7 mg l^-1, black bars) and a recovery period on hypothalamic <i>nNOS</i> mRNA levels (A) and plasma NO metabolites, nitrite plus nitrate (NOx) levels (B). Each value represents the mean±SEM (N = 7–11). Differences in the relative mRNA levels between the start of the experiment (CTL, control) and each treatment were tested by Dunnett’s test, *<i>p</i><0.05.</p

Protein carbonyl (PC) contents in Atlantic croaker exposed to environmental and laboratory hypoxia.

<p>(A) PC contents in croaker liver collected from normoxic (N1, N2) and hypoxic (F3, F4, C8, C9) sites in August 2007 (i), July 2008 (ii) and August 2012 (iii) in the nGoM. The horizontal lines represent mean values, N = 8. A nested ANOVA indicates PC contents in croaker liver from the normoxic sites were significantly different from those in fish from the hypoxic sites (**<i>p</i><0.01). Individual site differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters. DO, dissolved oxygen (mg l^-1). (B) Effects of 7-day laboratory exposure to normoxia (DO: ~6.5 mg l^-1, white bars), hypoxia (HYP, DO: 1.7 mg l^-1, black bars) and recovery period on PC contents in croaker liver. Each value represents the mean±SE (N = 7–8). Differences in the PC contents between the start of the experiment (CTL, control) and each treatment were tested by Dunnett’s test, *<i>p</i><0.05. prot., protein. (C) Proposed model of hypoxia-induced upregulation of croaker <i>hif-α</i>, <i>nNOS</i>, <i>igfbp</i> transcripts, NOx, and ROS and RNS (solid arrows pointing up), based on our present and previous studies (^1,2,3Rahman and Thomas, 2007, 2011, 2013). The model shows several pathways (dotted lines) through which hypoxia could potentially upregulate gene and biochemical biomarkers in croaker. ‘?’: evidence has only been obtained in mammalian <i>in vitro</i> studies (Li and Jackson, 2002).</p

Hierarchical clustering of gene expression in the brain and liver tissues of Atlantic croaker exposed to environmental and laboratory hypoxia.

<p>Croaker collected from normoxic (N1, N2) and hypoxic (C8, C9) sites in August 2007 and July 2008 in the nGoM (A) and laboratory hypoxia experiment (B). ^A: August; ^J: July.</p

Expression of <i>hif-1α</i> and <i>hif-2α</i> mRNA levels in brains of Atlantic croaker exposed to laboratory hypoxia.

<p>Effects of 7-day laboratory exposure to normoxia control (CTL, dissolved oxygen, DO: ~6.5 mg l^-1, white bars), hypoxia (HYP, DO: 1.7 mg l^-1, black bars) and a recovery period on relative <i>hif-1α</i> (A-ii) and <i>hif-2α</i> (B-ii) mRNA levels, and (C) C<i>t</i> values of <i>18S</i> rRNA in croaker brains. Note: here and in subsequent figures laboratory exposure duration only refers to period fish were exposed to target DO; fish were previously exposed to declining DO for additional 2-day adjustment period. After 7-day hypoxia exposure, DO of hypoxic treatment was restored to normoxic level (24-h recovery period). Each bar represents the mean±SEM (N = 7–8). Differences in the relative mRNA levels between the start of the experiment (CTL, control) and each treatment were tested by Dunnett’s test, *<i>p</i><0.05. ‘†’ indicates significant difference from normoxic controls (Student’s <i>t</i>-test, <i>p</i><0.05). C<i>t</i>, threshold cycle.</p

Brain <i>nNOS</i> mRNA and protein levels in Atlantic croaker collected from hypoxic and normoxic sites in the nGoM.

<p>Relative <i>nNOS</i> mRNA levels (A), (B) C<i>t</i> values of <i>18S</i> rRNA, and nNOS protein expression and levels (C,D) in brains of croaker collected from normoxic (N1, N2) and hypoxic (F3, F4, C8, C9) sites in August 2007 (i), July 2008 (ii), and August 2012 (iii). Representative Western blot of nNOS protein expression in croaker brain samples (B). The horizontal lines represent mean values, N = 8. A nested ANOVA indicates <i>nNOS</i> mRNA and protein levels in croaker from the normoxic sites were significantly different from those in fish from the hypoxic sites (**<i>p</i><0.01). Individual site differences identified with a multiple range test, Fisher's PLSD, are indicated with different letters. M, marker; kDa, kilodalton.</p