Lactation abolishes corticotropin-releasing factor-induced oxytocin secretion in the conscious rat

We have assessed the response of plasma oxytocin (OT) to intracerebroventricular CRF-41 in both virgin female and lactating rats. In virgin rats CRF-41 resulted in an increase in plasma OT from 5-30 min after administration. In lactating rats, however, there was a complete abolition of the OT response, even at the highest dose of CRF-41. These data demonstrate another feature of the hormone nonresponsiveness apparent during lactation and suggests that one of the reasons for the lack of stress responses could be a down-regulation of the response to endogenously released CRF-41.     

Insulin-induced hypoglycemia increases corticotropin-releasing factor messenger ribonucleic acid levels in rat hypothalamus

To study the effect of acute stress on CRF release and synthesis in rat hypothalamus, ACTH levels in plasma, CRF contents in the median eminence (ME), and CRF mRNA levels in the hypothalamus without ME and cerebral cortex were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min, while ME CRF content decreased at 30 and 60 min, then returned to the control level at 90 min. Hybridization with a cRNA probe revealed a single size class of CRF mRNA in the hypothalamus and cerebral cortex (approximately 1300 nucleotides), and the size of CRF mRNA in these tissues did not change during the experimental period. CRF mRNA levels in the hypothalamus increased to 130% of the control value at 30 min and reached a peak (186% of the control value) at 120 min, but these levels in the cerebral cortex did not change. These results suggest that insulin-induced hypoglycemia stimulates CRF synthesis by increasing CRF mRNA levels in the hypothalamus as well as CRF release, and that release and synthesis of CRF in the cerebral cortex are independent of those in the hypothalamus.     

Central administration of corticotropin-releasing factor modulates oxytocin secretion in the rat

Recently, it has been reported that oxytocin (OT), classically known for its function during parturition and lactation, is secreted in response to stressful stimuli in male rats. In these and in the present report it was found that swimming stress, restraint stress, ether stress, and footshock stress elevate OT secretion without affecting arginine-vasopressin (AVP) secretion. In the present studies, we investigated the possible modulation of OT secretion by CRF which is known to be released during stress. Male and female rats received intraventricular (icv) injections of 0.75 nmol (5 micrograms) rat CRF and were killed 5 min after the treatment. CRF significantly elevated OT secretion in male and female rats 3.4- and 4-fold, respectively. Plasma AVP levels were not affected by the treatment. The effect of CRF on OT release was structure specific since rat CRF, ovine CRF, and sauvagine were equipotent releasers of OT while an inactive analog to CRF, ovine CRF did not change plasma OT levels. In another set of experiments rats were pretreated with either CRF-antiserum (0.5 ml iv) or dexamethasone (20 micrograms/rat ip) and then injected with icv CRF. Both CRF-antiserum and dexamethasone blocked the rise in ACTH release after icv CRF completely but did not influence the OT response. This suggests CRF may be acting centrally but not at the level of the neurohypophysis to change OT secretion. Since parvocellular but not magnocellular neurons of the paraventricular nucleus have been demonstrated to be steroid sensitive in immunohistochemical studies, we suggest CRF may act directly or indirectly upon magnocellular neurons to increase OT release. Intravenous administration of 0.75 nmol CRF increased both OT and AVP levels in peripheral blood. The magnitude of this increase was similar (2- to 4-fold stimulation) to responses after icv administration of CRF. Intravenous administration of CRF results in hypotension and may therefore cause a baroreceptor mediated release of AVP and OT. From the above evidence we conclude: physical and mental stresses which do not result in changes in blood volume or osmolality evoke an increase in OT secretion while AVP secretion remains unchanged; CRF administered icv mimics OT responses observed after ether stress or footshock stress; CRF may play a role in regulating stress-induced OT secretion in the rat.     

Regulation of rat hypothalamic preprogrowth hormone-releasing factor messenger ribonucleic acid by dietary protein

To further evaluate nutrient regulation of GRF synthesis, we measured hypothalamic preproGRF messenger (m) RNA in food-deprived rats refed diets varying in nutrient composition by nuclease protection analysis. Adult male Sprague-Dawley rats were allowed free access to food (Fed), food deprived for 72 h (72-h FD), or 72 h FD then refed for 72 h with either a normal (NF) diet or isocaloric diets containing no protein (PF), carbohydrate (CF), or fat (FF). Seventy-two-hour FD rats displayed the expected 80% reduction in hypothalamic preproGRF mRNA. Upon refeeding, levels were normalized in rats refed NF, CF, or FF diets. In contrast, preproGRF mRNA in rats refed a PF diet was similar to that in 72-h FD rats. Rats refed a PF diet failed to gain weight and consumed less food than animals refed NF, CF, or FF diets. However, the lack of the GRF response to the PF diet was due to protein deprivation rather than caloric restriction, since hypothalamic preproGRF mRNA returned to 66% of Fed values in rats refed an equivalent amount (grams per day) of a NF diet. In 72-h FD rats refed isocaloric diets containing 4%, 8%, or 12% protein, preproGRF mRNA was restored to Fed values in a protein concentration-dependent manner being completely restored by the 12% diet. A lack of dietary protein was sufficient to regulate hypothalamic preproGRF mRNA since feeding rats a PF diet without prior food deprivation resulted in 70% reduction in preproGRF mRNA, whereas CF and FF diets were without effect. These data indicate that decreased hypothalamic preproGRF mRNA expression in 72-h FD rats occurs as a result of dietary protein deprivation.     

Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells

Angiogenesis is essential for endometrial growth and repair, and disruption of this process may lead to common disorders of women, including menorrhagia and endometriosis. In pregnancy, failure of the endometrial spiral arterioles to undergo remodeling leads to preeclampsia. Here we report that in addition to vascular endothelial growth factor A (VEGF-A), human endometrium expresses messenger ribonucleic acids (mRNAs) encoding VEGF-C, placenta growth factor (PlGF), the angiopoietins, angiopoietin 1 (Ang1) and Ang2, and the receptors VEGFR-3 (Flt-4), Tie 1, and Tie 2. Levels of VEGF-C, PlGF, and Tie 2 changed during the menstrual cycle. Intense hybridization for VEGF-C and PlGF mRNAs was found in uterine nature killer cells in secretory phase endometrium and for Ang2 mRNA in the same cells in the late secretory phase. Interleukin-2 (IL-2) and IL-15 up-regulated VEGF-C, but not PlGF or Ang2, mRNA levels in isolated NK cells. Conditioned medium from decidual NK cells did not induce human umbilical vein endothelial cell apoptosis. These results indicate that human endometrium expresses a wide range of angiogenic growth factors and that uterine nature killer cells may play an important role in the abnormal endometrial angiogenesis that underlies a range of disorders affecting women.     

Angiotensin II increases the corticotropin-releasing factor messenger ribonucleic acid level in the rat hypothalamus.

Angiotensin II (AII) has an important role in the regulation of CRF release. In the present study, the effect of centrally administered AII on CRF messenger RNA (mRNA) levels in the rat hypothalamus was examined. Administration of 0.1 nmol and 1 nmol AII into the lateral ventricle increased the levels of plasma ACTH 20 min and 45 min after administration and those of proopiomelanocortin mRNA in the anterior pituitary (AP) and CRF mRNA in the hypothalamus 2 h after administration. On the other hand, ACTH levels in AP and CRF levels in the median eminence temporarily decreased 45 min after the administration of 1 nmol AII, but it returned to the control level at 90 min. Administration of 10 nmol saralacin, an AII antagonist, blocked 1 nmol AII-induced increase in the levels of plasma ACTH, proopiomelanocortin mRNA in AP, and CRF mRNA in the hypothalamus. These results indicate that central administration of AII increases the CRF mRNA level in the hypothalamus in a receptor-specific manner and also increases CRF release. Therefore, AII seems to have an important role in the regulation of the release and synthesis of CRF in the hypothalamus.     

Assessment of corticotropin-releasing factor, vasopressin and somatostatin secretion by fetal hypothalamic neurons in culture

The concomitant release of corticotropin-releasing factor (CRF), vasopressin (AVP) and somatostatin (SRIF) has been followed from primary cultures of rat hypothalamic neurons. 18-day-old fetal rat hypothalami were dissociated enzymatically and mechanically, then plated and maintained in a serum-containing medium at a density of 2.5 x 10(6) cells per dish (equivalent to 3 hypothalami). Cultured neurons remained viable for up to 6 weeks, and peptide release was followed by immuno-assay between days 14 and 39 in culture. The incubation media were concentrated on C4 and C8 silica columns to facilitate detection of CRF and AVP. Peptide release was measured at various times up to 4 h, at which point it was still increasing. To optimise measurements, taking into account peptide degradation, a 1-hour incubation period was chosen for further studies. Release of CRF, AVP and SRIF by 56 mM K+ or 10 microM veratridine was statistically significantly greater than basal (p less than 0.01) and was Ca2-dependent. For CRF and AVP, stimulated release increased considerably with the age of culture, whereas SRIF release was steadier. Basal release for all 3 peptides did not fluctuate greatly over this period. Basal and stimulated release of the peptides continued over at least 5 successive 1-hour periods. At day 35 of culture, the peptide content was still increasing in a pattern which paralleled the increasing content in hypothalami freshly removed from age-matched rats. In conclusion, we have demonstrated a development of CRF, AVP and SRIF production by neurons over extended periods in culture as assessed by their peptide content and increasing responses to depolarizing stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of hypothalamic corticotropin-releasing hormone messenger ribonucleic acid (mRNA) precedes early-life experience-induced changes in hippocampal glucocorticoid receptor mRNA

Early-life experiences, including maternal interaction, profoundly influence hormonal stress responses during adulthood. In rats, daily handling during a critical neonatal period leads to a significant and permanent modulation of key molecules that govern hormonal secretion in response to stress. Thus, hippocampal glucocorticoid receptor (GR) expression is increased, whereas hypothalamic CRH-messenger RNA (mRNA) levels and stress-induced glucocorticoid release are reduced in adult rats handled early in life. Recent studies have highlighted the role of augmented maternal sensory input to handled rats as a key determinant of these changes. However, the molecular mechanisms, and particularly the critical, early events leading from enhanced sensory experience to long-lasting modulation of GR and CRH gene expression, remain largely unresolved. To elucidate the critical primary genes governing this molecular cascade, we determined the sequence of changes in GR-mRNA levels and in hypothalamic and amygdala CRH-mRNA expression at three developmental ages, and the temporal relationship between each of these changes and the emergence of reduced hormonal stress-responses. Down-regulation of hypothalamic CRH-mRNA levels in daily-handled rats was evident already by postnatal day 9, and was sustained through postnatal days 23 and 45, i.e. beyond puberty. In contrast, handling-related up-regulation of hippocampal GR-mRNA expression emerged subsequent to the 23rd postnatal day, i.e. much later than changes in hypothalamic CRH expression. The hormonal stress response of handled rats was reduced starting before postnatal day 23. These findings indicate that early, rapid, and persistent changes of hypothalamic CRH gene expression may play a critical role in the mechanism(s) by which early-life experience influences the hormonal stress-response long-term.     

Characterization of rat hypothalamic corticotropin-releasing factor.

A polypeptide was purified from rat hypothalamic extracts on the basis of its high intrinsic activity to release corticotropin (ACTH) from cultured rat anterior pituitary cells and its immunoactivity in a radioimmunoassay directed against the NH2 terminus (residues 4-20) of ovine hypothalamic corticotropin-releasing factor (CRF). Based on Edman degradation, peptide mapping, and amino acid analysis, the primary structure of this rat CRF was established to be: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. The hypophysiotropic potency of synthetic rat CRF did not deviate significantly from the potencies of the isolated native peptide or of synthetic ovine CRF. The close structural relationship between rat and ovine hypothalamic CRF is indicated by an 83% sequence homology.     

Central modulation of immunoreactive arginine vasopressin and oxytocin secretion into the hypophysial-portal circulation by corticotropin-releasing factor

Corticotropin-releasing factor (CRF), a potent ACTH secretagogue, has been found to exhibit many characteristics of central neurotransmitter/neuromodulatory substances. In this capacity, hypothalamic CRF might participate in postulated autoregulatory processes which regulate net secretion of adenohypophysical ACTH. We have examined the actions of centrally injected ovine CRF on the secretion of immunoreactive CRF, arginine vasopressin (AVP) and oxytocin (OT) into the hypophysial-portal circulation of urethane-anesthetized rats. Our observations do not support a short-term autoregulatory role for CRF. However, central administration of CRF was associated with a dose-dependent inhibition of hypophysial-portal concentrations of immunoreactive AVP and OT, suggesting potentially important central interactions among putative ACTH-regulatory factors.     

Bacterial lipopolysaccharide-induced coordinate downregulation of arginine vasopressin receptor V3 and corticotropin-releasing factor receptor 1 messenger ribonucleic acids in the anterior pituitary of endotoxemic steers

AVP and CRF are potent stimulators of pituitary ACTH secretion in cattle. Actions of AVP and CRF at the anterior pituitary are mediated by AVP receptor V3 (V3) and CRF receptor 1 (CRFR1). The primary objective of these studies was to determine the effect of systemic inflammatory stress on V3 and CRFR1 mRNAs in the bovine anterior pituitary. Holstein steers (n=20) were injected with 200 ng/kg bacterial lipopolysaccharide (LPS) and tissues collected 0, 2, 4, 12, and 24 h later. All animals responded to LPS administration with an increase in body temperature, plasma ACTH, and cortisol (p<0.05). Abundance of anterior pituitary V3 mRNA was decreased at 2, 4, and 12 h following LPS administration (p<0.05) and returned to basal by 24 h. A similar temporal regulation of pituitary CRFR1 mRNA (p<0.05), but not pituitary pro-opiomelanocortin (POMC) mRNA, was observed following LPS administration. Similar downregulation of CRFR1 mRNA was not observed in other brain regions following LPS administration (cerebellum, hypothalamus). Our results indicate that V3 and CRFR1 mRNAs are coordinately downregulated in the anterior pituitary during systemic inflammatory stress. Decreased AVP and CRF receptor expression may help regulate the pituitary-adrenal response to stress.     

Influence of the frequency of ovine corticotropin-releasing factor administration on adrenocorticotropin and corticosterone secretion in the rat

We have examined the pattern of ACTH secretion in rats receiving two or more injections of synthetic ovine corticotropin-releasing factor (CRF). A first exposure of cannulated intact rats to CRF caused a blunting of the ACTH response when CRF was again injected 0.5-6 h later. Since plasma corticosterone levels stayed elevated for only 2 h after the first injection of CRF, the diminished pituitary responsiveness persisted at a time when plasma corticosterone levels had returned to control values. When cannulated adrenalectomized animals were subjected to a similar treatment, the blunting of the ACTH release due to the second injection of CRF, though present, was of significantly shorter duration. Duplication of the corticosterone levels that follow one CRF injection by exogenous administration of the steroid inhibited the subsequent ACTH response to CRF for a maximum of 2 h. Finally, CRF infused into intact rats for 14 days consistently caused elevation of both ACTH and corticosterone secretion. We conclude that CRF administered in a repeated or continuous manner to intact rats, while causing some pituitary desensitization, still elicits a significant degree of ACTH secretion despite the presence of elevated corticosterone release. This observation as well as the fact that CRF-injected adrenalectomized animals exhibit some blunting of their ACTH responses suggest that steroid feedback, while participating in the diminished ability of CRF to repeatedly stimulate ACTH release, does not represent the sole modulator of pituitary responsiveness.     

Regulation of basal corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid expression in the paraventricular nucleus: effects of selective hypothalamic deafferentations

There exists considerable evidence to suggest that CRH and arginine vasopressin (AVP)-secreting parvocellular neurosecretory neurons of the hypothalamic paraventricular nucleus (PVN) are central integrators of negative feedback effects evoked by circulating glucocorticoid hormones. Most evidence suggests that these neurons may be receptive to circulating glucocorticoid levels, either via glucocorticoid receptors indigenous to these cells and/or via extrahypothalamic glucocorticoid-receptive neurons interacting with the PVN secretory cell. In an effort to address this issue, we performed anterior (ANT), posterior (POST) and total (TOT) deafferentations of the PVN region in male Sprague-Dawley rats using microknives fashioned from narrow-gauge spinal needles. Effective knife cuts were verified immunohistochemically, and deemed acceptable only if they avoided damage to the PVN proper and fibers of CRH and AVP-containing neurons coursing through the hypothalamus en route to the median eminence, while effectively eliminating neuronal input into the PVN region. Subsequent to surgery, levels of mRNA encoding for CRH and AVP in the parvocellular and magnocellular PVN were assayed via semiquantitative in situ hybridization histochemistry. Results indicate that TOT deafferentations resulted in significant increases in CRH mRNA expression in the PVN, and a slight but noticeable induction of AVP mRNA in the medial parvocellular but not posterior magnocellular divisions of the PVN. ANT lesions also produced an up-regulation of CRH and AVP mRNA relative to operated control rats. POST lesions did not produce a clear induction in either CRH or AVP mRNA. The data indicate that in the absence of neuronal input coming from anterior structures, CRH mRNA expression is up-regulated, suggesting that local effects of glucocorticoids on the PVN neuron are ineffective in maintaining normal CRH mRNA expression. These results support a role for neuronal feedback in regulation of the CRH neuron. The limited up-regulation (compared with adrenalectomized rats) of AVP mRNA in the TOT group suggests that while neuronal input may have some control of AVP mRNA expression, local glucocorticoid feedback is clearly able to restrict AVP message to levels considerably less than those seen in steroid-deficient animals. Analysis of knife-cut effects on plasma corticosterone and ACTH levels reveals that POST and TOT, but not ANT, deafferentations prohibit the secretory activity of the hypothalamo-pituitary-adrenocortical (HPA) axis seen pursuant to the anesthesia/thoracotomy in lesion and operated control groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Paradoxical responses of hypothalamic corticotropin-releasing factor (CRF) messenger ribonucleic acid (mRNA) and CRF-41 peptide and adenohypophysial proopiomelanocortin mRNA during chronic inflammatory stress.

We have determined the time course of the neuroendocrine response of Piebald-Viral-Glaxo (PVG) rats during the development of mycobacterially induced adjuvant arthritis. Anterior pituitary POMC mRNA increased at the time of onset of mycobacterially induced arthritis, but, paradoxically, coincident with the first signs of arthritis there was a consistent fall in CRF mRNA in the hypothalamic paraventricular nucleus. Coincident with this fall in CRF message, there was a corresponding decrease in CRF-41 peptide release into the hypophysial portal blood (HPB). In contrast, however, vasopressin release into the HPB was increased. There was an increase in adrenal weight associated with the development of arthritis, reflecting chronic activation of the HPA axis, which was reflected by increased circulating corticosterone concentrations. The synthetic adjuvant CP20961, which has different antigenic determinants, also caused an increase in POMC mRNA in the anterior pituitary, a decrease in CRF mRNA in the hypothalamic paraventricular nucleus, and a decrease in CRF-41 peptide release into the HPB in PVG rats 28 days after the induction of the arthritis. The arginine vasopressin level was not significantly different from the control value. In Sprague-Dawley rats, mycobacterial adjuvant resulted in a similar increase in POMC mRNA in the anterior pituitary 28 days after injection of the adjuvant. In this strain of rat there was no corresponding change in CRF mRNA. While there are some strain differences in the degree of change in CRF mRNA, both strains showed a common paradox of a marked increase in adenohypophyseal POMC mRNA not associated with increased CRF mRNA or peptide release. In the PVG strain of rat, CRF actually appears to be inhibited. The mechanisms involved in this disparity are unclear.     

Increased hypothalamic content of preproneuropeptide-Y messenger ribonucleic acid in streptozotocin-diabetic rats

Neuropeptide-Y (NPY) is a 36-amino acid C-terminally amidated peptide found within the hypothalamus that can potently stimulate carbohydrate feeding. Moreover, the hypothalamic content of NPY can be modulated by peripheral hetabolic status. To further evaluate the regulation of NPY synthesis in states of altered metabolic homeostasis, we measured the hypothalamic content of prepro-NPY mRNA in streptozocin (STZ)-diabetic, STZ-diabetic insulin-replaced, and control rats by both nuclease protection and in situ hybridization analyses. Adult male Sprague-Dawley rats received a single injection of STZ (100 mg/kg, ip) or citric acid (control). Beginning 72 h later one group of STZ-treated animals received daily injections of insulin (4 U Ultralente/day). All animals were killed 17-19 days after STZ or control treatment. STZ-treated animals were hyperglycernic and showed growth failure compared to control rats. Glycemic control was restored by insulin replacement, as was partial growth. Nuclease protection analysis revealed an approximately 3- to 4-fold increase in prepro-NPY mRNA levels in the samples from STZ-treated rats vs. control. This increase was returned to control values by insulin replacement. In situ hybridization analysis revealed the STZ-induced increase in hypothalamic prepro-NPY mRNA was detectable in the arcuate nucleus at levels that were in agreement with the nuclease protection results, but that NPY expression in other brain regions appeared to be either unaffected or decreased after STZ treatment. These data suggest that hypothalamic NPY expression is modulated by peripheral metabolic status and provide further explanation for the hyperphagia accompanying STZ-induced diabetes.     

In vivo corticotropin-releasing factor-induced secretion of adrenocorticotropin, beta-endorphin, and corticosterone

A 41-residue peptide purified as a corticotropin-releasing factor/beta-endorphin-releasing factor (CRF) in vitro was tested for its ability to stimulate the secretion of ACTH, beta-endorphin, and corticosterone in three animal groups: 1) unanesthesized rats bearing indwelling venous cannulae, 2) rats pretreated with chloropromazine plus morphine sulfate plus pentobarbital (CPZ-MS-Nb, and 3) rats with hypothalamic deafferentiations in the frontal and lateral retrochiasmatic areas. In all three bioassays iv administration of 0.1-10 micrograms CRF elicited a dose-related increase in plasma ACTH and beta-endorphin values over a 5- to 15-min period. Corticosterone secretion was also elevated but responded maximally with all doses of CRF tested. Pretreatment of CPZ-MS-Nb animals with 20 micrograms dexamethasone 4 h before assay abolished the CRF-induced hormone secretion. These data suggest that CRF may play a physiological role in the regulation of the hypothalamic-pituitary-adrenal axis.