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Endocrinology Vol. 139, No. 9 3822-3829
Copyright © 1998 by The Endocrine Society

ARTICLES

Peptide Gene Activation, Secretion, and Steroid Feedback during Stimulation of Rat Neuroendocrine Corticotropin-Releasing Hormone Neurons1

Susan M. Tanimura, Graciela Sanchez-Watts and Alan G. Watts

Program in Neural, Informational and Behavioral Sciences, and Neuroscience Graduate Program, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-2520

Address all correspondence and requests for reprints to: Alan G. Watts, D. Phil, Department of Biological Sciences, Hedco Neuroscience Building, MC 2520, University of Southern California, Los Angeles, California 90089-2520. E-mail: watts{at}rcf.usc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have used colloid-induced hypovolemia to investigate mechanisms operating in CRH neuroendocrine neurons of the hypothalamic paraventricular nucleus during a sustained stress. Specifically, three questions have been addressed using in situ hybridization and RIA. 1) Do neuropeptide secretion and gene activation share the same stimulus threshold? 2) Does corticosterone modulate mechanisms regulating CRH gene expression during sustained stress? 3) How are neuropeptides commonly colocalized with CRH affected? Our results show that the secretion of ACTH and activation of the CRH gene have distinct and separate stimulus thresholds. The threshold is higher for CRH gene activation than for ACTH secretion, suggesting some degree of mechanistic separation. In addition, corticosterone secreted during the first 3 h of sustained hypovolemia does not inhibit CRH gene expression. However, feedback inhibition may occur in the delayed time domain. Finally, neuropeptides colocalized with CRH are differentially regulated by sustained hypovolemia. Proenkephalin messenger RNA levels show a slower temporal response than those of CRH, while the vasopressin gene is not activated at any time in parvicellular neuroendocrine neurons. Our results emphasize that CRH neuroendocrine neurons respond to a stress event in a stimulus-specific manner in terms of both the profiles of secretion and gene expression, and the structure of glucocorticoid feedback.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RAT hypothalamo-pituitary-adrenal (HPA) axis is the principal motor system used by the brain to effect the neuroendocrine stress response. Its primary element is a pool of neuroendocrine neurons located in the dorsal aspect of the medial parvicellular subdivision of the hypothalamic paraventricular nucleus (PVHmpd). These neurons synthesize and release the ACTH secretogogues CRH and vasopressin (AVP) into hypophysial portal blood at a rate directly related to stimulus intensity (see Ref. 1 for review). ACTH then stimulates the synthesis and release of corticosterone from the adrenal cortex. In the unstimulated rat, corticosterone operates as a negative feedback signal to inhibit the synthesis and secretion of CRH, AVP, and ACTH. However, under stressed conditions, the operation of this regulatory mechanism becomes more complex (see Ref. 2 for review).

Stimulus/response coupling in CRH neuroendocrine neurons during stress involves at least four processes: stimulus onset and transduction, initiation of secretogogue (neuropeptide) release, activation of neuropeptide gene expression, and feedback regulation. The temporal organization and functional interactions between these components are currently poorly defined in CRH neuroendocrine neurons. To help clarify how these elements interact during a sustained stress event, we report here the temporal profiles for a number of secretory and synthetic processes associated with the CRH neuroendocrine neuron during sustained hypovolemia obtained from a single set of animals. Examining the structure of these profiles allows us to address the following questions. First, how are peptidergic gene activation and secretory events related in the CRH neuron? Second, does corticosterone act as an inhibitory signal for CRH gene expression in the PVHmpd during periods of sustained secretion? And finally, what are the concurrent effects of this stressor on neuropeptide genes colocalized with CRH?

We have chosen sustained hypovolemia as a stimulus because it has a physiological onset identifiable with some precision, its intensity is quantifiable, and it is maintained for some hours (3). Furthermore, it is an exclusively viscerosensory stimulus, at least in its early phase, and reduces extracellular fluid volume to trigger a set of very well defined behavioral and neuroendocrine responses aimed at restoring fluid homeostasis (4). Sustained hypovolemia is induced experimentally by sc injections of polyethylene glycol (PEG) that continually sequester isotonic, protein-free extracellular fluid into a biologically inaccessible edema. PEG injections increase plasma corticosterone and renin concentrations (5) as well as PVHmpd levels of CRH, proenkephalin (pENK), and neurotensin messenger RNAs (mRNAs) (6), although the time course of these events is currently unknown.

With these attributes in mind, we describe the temporal profile of the secretory and synthetic responses to sustained hypovolemia using data generated every hour for the first 6 h after PEG injections given during the early part of the light period. We measured plasma concentrations of ACTH, corticosterone, and PRL to address the secretory response. In these same animals we measured levels of CRH, c-fos, pENK mRNAs, and the primary transcripts of CRH and AVP in the PVHmpd using in situ hybridization to determine the synthetic responses.

Some of these data have been presented in abstract form (7, 8).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adult male Sprague-Dawley rats (225–250 g BW at the beginning of the experiment) were housed three per cage and maintained on a 12-h light, 12-h dark photoperiod (lights on at 0600 h) with unlimited water and rat chow and allowed at least 5 days of acclimation to animal quarters. Six animals were assigned for each treatment group at every time point with the exception of the 0- and 2-h PEG-treated groups, in which 12 animals were used.

PEG injection
Under brief halothane anesthesia, 5-ml sc injections of either 40% (wt/vol) PEG (MW 8000, Sigma Chemical Co., St. Louis MO) dissolved in 0.9% saline or 0.9% saline at room temperature were performed between 0700–0800 h. Each injection took less than 3 min from the onset of anesthesia to recovery of consciousness. At this time, all water bottles and food were removed from the cages, and animals were left undisturbed until death.

Decapitation and tissue handling
Animals were killed by decapitation at assigned time points between 0800–1300 h. Trunk blood was collected in two cooled vials, coated with either EDTA-saline for ACTH assay or heparin-saline for corticosterone and PRL assays. Hematocrits were measured, and plasma was separated and stored at -20 C for plasma ACTH, corticosterone, and PRL determinations at a later date. Plasma volume deficit was derived from hematocrit ((hemactocritPEG - hematocritmean control)/ hematocritPEG) x 100.

Brains were rapidly removed and fixed by immersion in ice-cold 4% paraformaldehyde in 0.1 M borate buffer, pH 9.5, overnight. Sucrose was added to the 4% paraformaldehyde solution to attain a 12% sucrose concentration and then fixed for 2 additional days. Brains were frozen in powdered dry ice and immediately stored at -70 C until sectioning at a later date. Eight series of one in eight 15-µm thick coronal sections were cut through the rostral hypothalamus and saved in ice-cold potassium PBS containing 0.25% paraformaldehyde. The sections were mounted the same day on poly-L-lysine-coated gelatin-subbed slides, vacuum desiccated overnight, postfixed in KPBS-4% paraformaldehyde for 1 h at room temperature, rinsed five times for 5 min each time in clean KPBS, air-dried, and then stored at -70 C in air-tight containers containing silica gel desiccant for hybridization at a later date. Serial sections were saved for thionin staining.

In situ hybridization
Serial sections through the rostral hypothalamus were each hybridized with [35S]UTP-labeled complementary RNA (cRNA) probes transcribed using the Promega Gemini kit (Promega, Madison, WI), with appropriate RNA polymerases, from a 700-bp complementary DNA (cDNA) sequence coding for part of the mRNA encoding prepro-CRH, a 935-bp cDNA sequence for the entire coding sequence of preproenkephalin, a 536-bp PvuII fragment complementary to the sequence within the single CRH intron, a complement to a 2.1-kb region of the cDNA sequence coding for c-fos, or a complementary sequence to the 700-bp PvuII fragment of intron 1 of the AVP gene. Control hybridization experiments for each probe have been previously described (6, 9).

In situ hybridization with the 35S-labeled cRNA probes was performed as previously described (6), with posthybridization modifications for CRH heteronuclear (hn) RNA as follows. After the ribonuclease incubation and room temperature washes from 4 to 0.1 x SSC, slides were incubated at 70 C for 30 min with slight agitation every 10 min. Sections were exposed to Cronex Microvision x-ray film (DuPont, Wilmington, DE) for appropriate exposure periods (4–21 days), then dipped in nuclear track emulsion (Kodak NTB-2, diluted 1:1 with distilled water), exposed for 5–25 days, developed, and counterstained with thionin.

RIA
Plasma corticosterone, PRL, and ACTH were measured in duplicate unextracted samples by double antibody RIAs. Plasma ACTH concentrations were determined using an [125I]ACTH double antibody RIA as previously described (10). The lower sensitivity was 20 pg/ml, and the intraassay coefficient of variation was 1.8%. Plasma corticosterone concentrations were determined as described previously (11) using an [125I]corticosterone double antibody RIA supplied in kit form (ICN Biochemicals, Costa Mesa, CA). The lower sensitivity limit was 15 ng/ml, and the intraassay coefficient of variation was less than 8%. Plasma PRL concentrations were determined by double antibody RIA as previously described (12). PRL RP-3 (NIAMDD) was used as the reference standard. The lower sensitivity limit was 1.0 ng/ml, and the intraassay coefficient of variation was 6.1%. All samples were measured in single assays.

Quantitation of [35S]UTP cRNA hybridization
Mean gray levels (MGL) of mRNA hybridization signals in the Nissl-defined PVHmpd were measured from images on Cronex microvision x-ray film as described by Watts and Sanchez Watts (6).

A preliminary experiment was performed to verify the sensitivity of the CRH hnRNA hybridization analysis. Six animals were placed under ether anesthesia for 5 min, which has been reported to significantly increase CRH hnRNA levels in the PVH (13). Rats were placed singly in a glass chamber saturated with ether vapor. Once anesthetized, animals were removed from the chamber and exposed to ether-soaked cotton wool applied to the nose as necessary. Animals were maintained under ether anesthesia for 5 min and then perfused. Four control rats were injected ip with tribromoethanol and immediately perfused. Sections were processed for CRH hnRNA in situ hybridization as described above.

Levels of CRH and AVP hnRNA hybridization in the PVHmpd were determined using the method of Kovács and Sawchenko (13). Here, the total number of nuclei with numbers of silver grains 5 times greater than background was counted in three 1:8 sections centered on the PVHmpd using the adjacent Nissl-stained and CRH-hybridized sections as reference.

Statistical analysis
The significance of differences in dependent variables between euhydrated and PEG-treated animals across the experiment were determined using single factor ANOVA, followed by Dunnett’s two-tailed post-hoc test (with 0 h values taken as the control) or Tukey post-hoc test. The significance of differences between saline- and PEG-injected animals at individual times was determined using Student’s t test. P < 0.05 was regarded as being statistically significant for all tests. All statistical analyses were performed using Excel (Mac version 4.0; Microsoft, Redmond, WA) and Systat (Mac version 5.2, Systat, Evanston, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hematocrit
Figure 1AGo shows that a significant increase in hematocrit first occurred 1 h after PEG injection (P < 0.05) compared with the level in saline-injected animals and was maintained in all PEG-injected animals for the duration of the experiment (P < 0.002–0.0001). The hematocrit did not significantly increase at any time in saline-injected controls.



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  		Figure 1. A, Mean (+SEM) hematocrits and equivalent plasma volume deficit; plasma ACTH (B) and corticosterone (C) concentrations in rats injected sc with either 5 ml vehicle (0.9% saline; open bars) or 40% PEG (black bars). See text for level of significance. D, Individual plasma ACTH concentration of all animals as a function of plasma volume deficit. The vertical dashed line depicts the plasma volume deficit threshold necessary to elicit an ACTH response.

 
Secretory response
Plasma ACTH. Figure 1BGo shows that a significant increase in the plasma ACTH concentration occurred 2 h after PEG injection (P < 0.01) compared with the level in saline-injected animals at 0 h. Elevated levels were maintained in all PEG-injected animals for the duration of the experiment (P < 0.05–0.001). Plasma ACTH concentrations did not significantly increase at any time in saline-injected controls. At 6 h, plasma ACTH concentrations remained significantly increased (P < 0.001) in hypovolemic rats. However, they tended to decrease compared with values at 5 h, although this was not statistically significant.

Plasma corticosterone and PRL. Plasma corticosterone concentrations increased in parallel with ACTH levels in hypovolemic rats (Fig. 1CGo) and were significantly increased above levels in saline-injected animals at all time points from 2 h onward (P < 0.01 to P < 0.0001). A stimulus-associated plasma PRL response did not occur at any time in hypovolemic rats; plasma PRL concentrations did not significantly increase above levels in saline-injected rats; the mean range for all groups during the entire experiment was between 1.9 ± 0.2 and 5.6 ± 1.7 ng/ml.

Plasma volume deficit and ACTH response. Figure 1DGo shows that significantly elevated ACTH secretion only occurred when a plasma deficit threshold of about 12% was exceeded. Figure 1Go, A and B, shows that this threshold was only crossed in PEG-injected animals sometime between 1–2 h when significant increases in plasma ACTH were first measured (P < 0.01).

Synthetic response
CRH hnRNA. In the preliminary experiment using 5 min of ether exposure, we measured a robust increase in the number of CRH hnRNA-labeled cells (138.0 ± 9.0) in the PVHmpd compared with that in nonexposed controls.

The signal from the CRH mRNA hybridization was localized over the cytoplasm of neurons (Fig. 2AGo), whereas that for CRH hnRNA was localized over the neuronal nucleus (Fig. 2BGo). The first significant increase in the number of CRH hnRNA-positive cells was seen 3 h after PEG injection (Fig. 3AGo; P < 0.01). The number of labeled cells in PEG-injected animals then remained significantly elevated above the control value until the end of the experiment (P < 0.05–0.0001).



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  		Figure 2. Brightfield photomicrographs of hybridization for CRH mRNA (A) and CRH hnRNA (B) on sections counterstained with thionin to show cell nuclei. Note the cytoplasmic labeling for the mRNA and the nuclear labeling for the hnRNA (scale bar = 5 µm).

 


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  		Figure 3. Mean (+SEM) of A) the number of CRH hnRNA-labeled cells in rats injected sc with 5 ml of either vehicle (0.9% saline; open bars) or 40% PEG (black bars) as a function of time after injection, and B) individual CRH hnRNA cell counts plotted as a function of plasma volume deficit. The vertical dashed line signifies the plasma volume deficit threshold necessary to induce CRH gene transcription (CRH hnRNA). C, Mean (±SEM) MGL of CRH mRNA hybridization in the PVHmpd expressed in arbitrary units of animals injected sc with 5 ml of either vehicle (0.9% saline; open bars) or 40% PEG (black bars) as a function of time after injection. See text for level of significance.

 
Plasma volume deficit and CRH gene transcription. CRH gene transcription had a higher response threshold to hypovolemic stress (Fig. 3BGo) than the response of ACTH secretion (Fig. 2DGo); CRH hnRNA levels did not increase until the plasma volume deficit was above approximately 20%. This occurred between 2–3 h after PEG injection (Fig. 3AGo).

CRH mRNA. The content of CRH mRNA in the PVHmpd of saline-injected animals (Fig. 3CGo) gradually declined from 0 until 5 h, so that values at 3 h (P < 0.01) and 5 h (P < 0.001) were significantly lower than those at 0 h. This decline was prevented in PEG-injected animals; CRH mRNA levels were significantly greater at 3 h (P < 0.01), 4 h (P < 0.001), and 5 h (P < 0.001) after injection than in saline-injected animals at 3 and 5 h. However, between 5 and 6 h, the CRH mRNA content was sharply reduced in PEG-injected animals to levels that were no longer significantly different from those seen 5 h after injection with saline.

AVP hnRNA. Levels of AVP hnRNA remained very low in all saline-injected animals. At no time were these significantly increased in any animals injected with PEG compared with those in animals injected with saline (positively labeled cells in the PVHmpd at 5 h: saline, 4.0 ± 1.0; PEG, 3.0 ± 1.0).

c-fos. Figure 4AGo shows that levels of c-fos mRNA in PVHmpd were very low at 0 h, but were significantly increased in both saline-treated (P < 0.0002) and PEG-treated (P < 0.005) groups 1 h after injection, most likely because of the brief halothane anesthesia and surgery used for the injections. At 2 h, c-fos mRNA had returned to levels not significantly different from those in 0 h controls in both treatment groups. The first significant increase in c-fos mRNA in the PVHmpd compared with saline-injected controls was measured at 3 h in hypovolemic rats (P < 0.05). Levels continued to increase from 3 h onward, remaining elevated in all PEG-injected animals for the duration of the experiment (P < 0.005–0.0001). Although mean levels in PEG-injected animals were lower at 6 h compared with those at 5 h, this was not statistically significant.



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  		Figure 4. Mean (+SEM) MGL of c-fos (A) and pENK mRNA (B) hybridizations in the PVHmpd after sc injection of either vehicle (0.9% saline; open bars) or 40% PEG (black bars) expressed in arbitrary units. See text for level of significance.

 
pENK mRNA. Figure 4BGo shows that the pENK mRNA response in the PVHmpd to hypovolemia was delayed compared with those of CRH and c-fos mRNAs. Significant increases in pENK mRNA in hypovolemic rats were first detectable after 4 h compared with those in 0 h (P < 0.005) and 3 h saline-injected controls (P < 0.01). These elevated levels were maintained until the end of the experiment (5 h, P < 0.0005; 6 h, P < 0.0005; vs. saline-treated group at 5 h).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that the sustained hypovolemia evoked by sc PEG injections is accompanied by a specific, sequential, and coordinate response from the motor elements of the HPA axis (Fig. 5Go). The physiological structure of this response combined with our ability to measure the levels of neuropeptide primary transcripts, mRNAs, and plasma hormone concentrations in the same animals allows us to address three central issues concerning the dynamics of secretion, gene expression, and feedback regulation in neuroendocrine motor neurons: 1) the relationship between the onsets of secretion and gene activation, 2) the effects of corticosterone feedback, and 3) concurrent effects of the stressor on neuropeptides colocalized with CRH.



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  		Figure 5. Schematic summary of the temporal response observed along the HPA axis as sustained hypovolemia progresses. The hatched bar along the c-fos mRNA response sequence denotes the increase in mRNA levels observed in both saline- and PEG-injected animals and is attributed to the nonspecific stress of anesthesia and sc injection. Based upon the indexes measured, ACTH and CORT secretion (open bars) precede CRH gene transcription (CRH hnRNA) and increases in c-fos, CRH, and pENK mRNA levels (black bars), with no AVP gene response (AVP hnRNA) measured (see text for details).

 
However, before discussing the implications of our findings it is worthwhile considering two points relating to the character of the stressor we have used. First, we have shown that sustained hypovolemia differs from ether anesthesia, restraint, or immobilization in that it is not accompanied by increased PRL secretion (14, 15, 16). This observation shows that sustained hypovolemia activates a different set of afferents to the neuroendocrine PVH than these other stressors. Second, sustained hypovolemia is a hemodynamic stressor that, in terms of sensory signaling, has some similarities to hypotension and hemorrhage. In this context three sets of studies in the rat suggest that CRH rather than AVP is the major ACTH secretogogue associated with these other hemodynamic stressors. First, ACTH secretion after hemorrhage is abolished by lesions of the PVH (17) or by immunoneutralization of CRH (18). Second, hemorrhage or nitroprusside-induced hypotension induces CRH release into hypophysial portal blood, whereas there is no detectable rise in AVP concentrations after nitroprusside-induced hypotension (19, 20). Finally, hemorrhage or sustained hypovolemia increases CRH, pENK, and neurotensin, but not AVP mRNAs, in the PVHmpd (6, 21).

Relationship between the onset of secretion and the onset of gene activation
In models where the stressor can be quantified, a relatively simple relationship exists between the intensity of stress and the secretory response of neuroendocrine motor neurons. In particular, a tight correlation has been shown between hemorrhage and ACTH secretion (22, 23), and for oxytocin and vasopressin secretion to sustained hypovolemia (24, 25). Our data now show a similar stimulus-secretion correlation between sustained hypovolemia and ACTH secretion. Furthermore, like other sensori-motor interactions, significant ACTH secretion only occurs once a physiologically identifiable threshold is crossed; in this experiment, a 12% plasma volume deficit, which occurs between 1–2 h after injection.

Identifying the onset of ACTH secretion is currently the only way to relate the onset of secretogogue release with those processes regulating CRH synthesis. This is because methods that provide direct estimates of ACTH secretogogue release into hypophysial portal blood depend upon prolonged anesthesia and surgery (10, 19), which would undoubtedly activate genes nonspecifically in the PVH. Considering this caveat, establishing that the onset of stimulus-dependent CRH release occurs no later than 2 h after PEG injection allows us to relate the timing of this event to those of others occurring in CRH neurons.

Two hours after PEG injection, we show that although the CRH neuron is actively releasing secretogogue into hypophysial portal blood (as indicated by elevated plasma ACTH concentrations), there is no measurable increase in either the CRH or AVP primary transcripts in the PVHmpd. Increased transcription of the CRH gene above that of controls was first seen at least 1 h after significant increases in ACTH secretion had occurred. In this context, it is important to consider three points. First, our results cannot be explained by the presence of a fixed lag period between the onset of gene activation and the production of the primary transcript, as would be required for new protein synthesis; CRH hnRNA levels increase as rapidly as 5 min of stimulus onset (9, 13, 26). Second, significantly elevated levels of CRH hnRNA can still be measured up to 1 h after a transient stressor (13), indicating that the half-life of the hnRNA is long enough to detect significant increases at each of our time points. Finally, preliminary experiments show that our assay system was sufficiently sensitive to detect increases in CRH hnRNA 5 min after ether anesthesia, demonstrating that we are not missing a significant period of activated transcription. Therefore, these data show that stimulus-induced CRH release and subsequent gene activation possess distinct and separate thresholds, suggesting some degree of mechanistic dissociation.

The accumulation rate of CRH mRNA in the PVHmpd of unstimulated animals is not constant over a 24-h period (2), and any perturbation to the HPA axis is superimposed upon this circadian pattern (27). Thus, a decline in CRH mRNA levels, consistent with the circadian pattern of CRH mRNA accumulation (28, 29), was seen in our saline-injected animals. However, when the sustained stressor was presented, this decline was abolished, so that CRH mRNA levels were significantly higher than equivalently timed control values 3, 4, and 5 h after injection. Taken together with the CRH hnRNA data from the same animals, the prevention of this decline can be accounted for at least in part by steadily increased CRH gene transcription.

Effects of corticosterone feedback
At least three sets of afferent inputs convey sensory information to CRH neuronal cell bodies and terminals at the median eminence during sustained hypovolemia: 1) decreased output from low pressure baroreceptor signal reductions in blood volume through the vagus and glossopharyngeal nerves to the nucleus of the solitary tract. The nucleus of the solitary tract along with other brain stem catecholaminergic afferents sends this information rostrally to the PVH (30, 31). Second, increased plasma concentrations of angiotensin II access the brain through AII receptors in the subfornical organ, which, in turn, projects to the PVH.

The third afferent signal important for regulating the size of the CRH mRNA pool is circulating corticosterone (see Ref. 2 for review). Keller-Wood and Dallman (32) proposed that the negative feedback action of corticosterone acts on ACTH release in three time domains, rapid, delayed, and slow, and it is valuable to use this same concept when considering how corticosterone regulates the CRH gene. Here we show that levels of CRH hnRNA, mRNA, and plasma ACTH all remain elevated above control values for at least 3 h despite sustained corticosterone secretion. This suggests that negative feedback actions in the rapid and perhaps the early delayed phase either do not operate or are overridden. This conclusion is consistent with our recent studies (33); we saw no significant difference between the size of the CRH mRNA response to PEG at 5 h in intact animals able to mount a robust corticosterone secretory response to hypovolemia and that in adrenalectomized animals given a low dose corticosterone replacement (sufficient in normalize thymus weights) but incapable of secreting corticosterone.

Our data showing that increasing plasma corticosterone concentrations do not significantly inhibit ACTH secretion for at least 5 h after adrenocortical activation has occurred are intriguingly similar to those of Keller-Wood and Dallman (32). These workers pointed out that there were two types of stressor with respect to how corticosterone rapid feedback impacts ACTH secretion: corticosterone-sensitive (e.g. ether anesthesia) and corticosterone-insensitive (e.g. hemorrhage). Our results now show that a similar difference may occur regarding corticosterone feedback action on CRH gene regulation and make the critical point that feedback mechanisms operating during one stressor may not act on CRH gene expression in the same manner as those during another.

The rather abrupt and significant reduction in CRH mRNA seen between 5 and 6 h is striking and may reflect a corticosterone feedback inhibitory component acting in the delayed time domain, particularly as it occurred at a time when ACTH secretion was also beginning to decline. This decline in mRNA seems unlikely to have resulted from nonspecific influences on CRH neurons because pENK and c-fos mRNAs, mRNAs previously shown to be colocalized with CRH in these circumstances (6, 13), as well as CRH hnRNA were all still significantly elevated at this time. One explanation for the reduction in CRH mRNA is that corticosterone is interacting with mechanisms responsible for processing of cytoplasmic mRNAs. Thus, at this point of the stress event, either corticosterone begins to inhibit afferent signaling to CRH neurons (perhaps as reflected in the downward trend in CRH hnRNA and c-fos mRNA levels seen at this time), or CRH mRNA stability is compromised. Iredale and Duman (34) recently showed that corticosterone reduced CRH-R1 receptor mRNA stability by 50% in pituitary-derived AtT-20 cells by a process dependent upon de novo protein synthesis; a similar mechanism may operate as the stress event progresses.

Concurrent effects of the stressor on neuropeptides colocalized with CRH
The fact that AVP gene transcription did not increase at any time during the response to sustained hypovolemia confirms our previous results with colocalized neuropeptide mRNAs (6). They are consistent with the idea that, as in other hemodynamic stressors, AVP does not play a role in activating the HPA axis in this model. Interestingly, elevated levels of pENK mRNA were not seen until 1 h after the CRH gene had been activated. This suggests that there is significant divergence in the signal transduction mechanisms regulating pENK, CRH, and AVP genes. Those responsible for controlling pENK mRNA may require a higher stimulus threshold or a longer synthesis period than the CRH gene, as is the case for the AVP gene (13). However, it should be noted that we are measuring levels of pENK mRNA and not the primary transcript, and it is possible that activations of both CRH and pENK occur simultaneously, but at this time of day, significant time is required for measurable amounts of pENK to become detectable. Taken together these data are consistent with the idea that distinct cellular signaling pathways control the AVP, pENK, and CRH genes during stress.


   Acknowledgments
 
We are very grateful to John Bennie, Sheena Carroll, and George Fink of the Medical Research Council Brain Metabolism Unit (Edinburgh, Scotland) for the ACTH and PRL assays, and to Drs. Tom Curran, Joseph Majzoub, Steven Sabol, Thomas Sherman, and Robert Thompson for the cDNAs used to generate the riboprobes.


   Footnotes
 
1 This work was supported by Grants NS-29728 and KO-4–01833 (to A.G.W.) from the NINDS, NIH. Back

Received March 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Whitnall MH 1993 Regulation of the hypothalamic corticotropin-releasing hormone neurosecretory system. Prog Neurobiol 40:573–629[Medline]
  2. Watts AG 1996 The impact of physiological stimuli on the expression of corticotropin-releasing hormone (CRH) and other neuropeptide genes. Front Neuroendocrinol 17:281–326[CrossRef][Medline]
  3. Stricker EM, Jaloweic JE 1970 Restoration of intravascular fluid volume following acute hypovolemia in rats. Am J Physiol 218:191–196[Free Full Text]
  4. Stricker EM 1981 Thirst and sodium appetite after colloidal treatment in rats. J Comp Physiol Psychol 95:1–25[CrossRef][Medline]
  5. Stricker EM, Vagnucci AH, McDonald RH, Leenan FH 1979 Renin and aldosterone secretions during hypovolemia in rats: relation to NaCl intake. Am J Physiol 237:R45–R51
  6. Watts AG, Sanchez-Watts G 1995 Physiological regulation of peptide messenger RNA colocalization in rat hypothalamic paraventricular medial parvicellular neurons. J Comp Neurol 352:501–514[CrossRef][Medline]
  7. Tanimura SM, Sanchez-Watts G, Watts AG Corticosterone modulates corticotropin-releasing hormone gene expression associated with hypovolemia in the paraventricular nucleus of the hypothalamus. 26th Annual Meeting of the Society for Neuroscience, Washington, DC, 1996, p 335.9 (Abstract)
  8. Tanimura SM, Watts AG Corticosterone facilitates as well as inhibits CRH gene expression in the rat hypothalamic paraventricular nucleus. 27th Annual Meeting of the Society for Neuroscience, New Orleans, LA, 1997, p 392.4 (Abstract)
  9. Herman JP, Schafer MK, Thompson RC, Watson SJ 1992 Rapid regulation of corticotropin-releasing hormone gene transcription in vivo. Mol Endocrinol 6:1061–1069[Abstract]
  10. Fink G, Robinson ICAR, Tannahill LA 1988 Effect of adrenalectomy and glucocorticoids on the peptides CRF-41, AVP and oxytocin in rat hypophysial portal blood. J Physiol 401:329–346[Abstract/Free Full Text]
  11. Watts AG, Sanchez-Watts G 1995 Region-specific regulation of neuropeptide mRNAs in rat limbic forebrain neurones by aldosterone and corticosterone. J Physiol 484.3:721–736
  12. Watts AG, Sheward WJ, Whale D, Fink G 1989 The effects of knife cuts in the subparaventricular zone of the female rat hypothalamus on oestrogen-induced diurnal wheel-running activity. J Endocrinol 122:593–604[Abstract/Free Full Text]
  13. Kovacs KJ, Sawchenko PE 1996 Sequence of stress-induced alterations in indices of synaptic and transcriptional activation in parvocellular neurosecretory neurons. J Neurosci 14:4825–4830[Abstract]
  14. Minamitani M, Minamitani T, Lechan RM, Bollinger-Gruber J, Reichlin S 1987 Paraventricular nucleus mediates prolactin secretory responses to restraint stress, ether stress, and 5-hydroxy-L-tryptophan injection in the rat. Endocrinology 120:860–867[Abstract]
  15. Gray TS, Piechowski RA, Yracheta JM, Rittenhouse PA, Bethea CL, Van de Kar LD 1993 Ibotenic acid lesions in the bed nucleus of the stria terminalis attenuate conditioned stress-induced increases in prolactin, ACTH and corticosterone. Neuroendocrinology 57:517–524[Medline]
  16. Larsen PJ, Mau SE 1994 Effect of acute stress on the expression of hypothalamic messenger ribonucleic acids encoding the endogenous opiod precursors proenkephalin-A and proopiomelanocortin. Peptides 15:783–790[CrossRef][Medline]
  17. Darlington DN, Shinsako J, Dallman MF 1988 Paraventricular lesions: hormonal and cardiovascular responses to hemorrhage. Brain Res 439:289–301[CrossRef][Medline]
  18. Plotsky PM, Bruhn TO, Vale W 1985 Evidence for multifactorial regulation of adrenocorticotropin secretory response to hemodynamic stimuli. Endocrinology 116:633–639[Abstract]
  19. Plotsky PM, Vale W 1984 Hemorrhage-induced secretion of corticotropin-releasing factor-like immunoreactivity into the rat hypophysial portal circulation and its inhibition by glucocorticoids. Endocrinology 114:164–169[Abstract]
  20. Plotsky PM, Otto S, Sapolsky RM 1986 Inhibition of immunoreactive corticotropin-releasing factor secretion into the hypophysial-portal circulation by delayed glucocorticoid feedback. Endocrinology 119:1126–1130[Abstract]
  21. Darlington DN, Barraclough CA, Gann DS 1992 Hypotensive hemorrhage elevates corticotropin-releasing hormone messenger ribonucleic acid (mRNA) but no vasopressin mRNA in the rat hypothalamus. Endocrinology 130:1280–1288
  22. Gann DS, Ward DG, Carlson DE 1978 Neural control of ACTH: a homeostatic reflex. Recent Prog Horm Res 34:357–400
  23. Darlington DN, Shinsako J, Dallman MF 1986 Responses of ACTH, epinephrine, norepinephrine, and cardiovascular system to hemorrhage. Am J Physiol 251:H612–H618
  24. Dunn FL, Brennan RJ, Nelson AE, Robertson GL 1973 The role of blood osmolality and volume in regulating vasopressin secretion in the rat. J Clin Invest 401:329–346
  25. Stricker EM Verbalis JG 1986 Interaction of osmotic and volume stimuli in regulation of neurohypophyseal secretion in rats. Am J Physiol 250:R267–R275
  26. Imaki T, Xiao-Quan W, Shibasaki T, Yamada K, Harada S, Chikada N, Naruse M, Demura H 1995 Stress induced activation of neuronal activity and corticotropin-releasing factor gene expression in the paraventricular nucleus is modulated by glucocorticoids in rats. J Clin Invest 99:231–238
  27. Dallman MF, Akana SF, Cascio CS, Darlington, DN, Jacobson L, Levin N 1987 Regulation of ACTH secretion: variations on a theme of B. Recent Prog Horm Res 43:113–173
  28. Watts AG, Swanson LW 1989 Diurnal variations in the content of preprocorticotropin-releasing hormone messenger ribonucleic acids in the hypothalamic paraventricular nucleus of rats of both sexes as measured by in situ hybridization. Endocrinology 125:1734–1738[Abstract]
  29. Cai A, Wise PM 1996 Age related changes in the diurnal rhythm of CRH gene expression in the paraventricular nuclei. Am J Physiol 270:E238–E243
  30. Sawchenko PJ, Swanson LW 1981 Central noradrenergic pathways for the integration of hypothalamic neuroendocrine and autonomic responses. Science 214:685–687[Abstract/Free Full Text]
  31. Cunningham Jr ET, Sawchenko PE 1988 Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat. J Comp Neurol 274:60–76[CrossRef][Medline]
  32. Keller-Wood ME, Dallman MF 1984 Corticosteroid inhibition of ACTH secretion. Endocr Rev 5:1–24[CrossRef][Medline]
  33. Tanimura SM, Watts AG 1998 Corticosterone can facilitate as well as inhibit CRH gene expression in the rat hypothalamic paraventricular nucleus. Endocinology 139:3830–3836[Abstract/Free Full Text]
  34. Iredale PA, Duman RS 1997 Glucocorticoid regulation of corticotropin-releasing factor (1) receptor expression in pituitary-derived AtT-20 cells. Mol Pharmacol 51:794–799[Abstract/Free Full Text]




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