| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Pharmacological and Physiological Science, Saint Louis University, St. Louis, Missouri 63104
Address all correspondence and requests for reprints to: Meghan M. Taylor, Saint Louis University, Department of Pharmacological and Physiological Science, 1402 South Grand Boulevard, St. Louis, Missouri 63104. E-mail: taylormm{at}slu.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In vivo experiments
Under ketamine (Ketaset, Fort Dodge Animal Health, Fort Dodge, IA)/xylazine (TranquiVed, Vedco Inc., St. Joseph, MO) anesthesia (60 mg/8 mg mixture·ml, 0.1 ml/100 g body weight, ip injection), rats were placed in a stereotaxic device and a 23-gauge, stainless steel cannula (17 mm) was implanted into the right lateral cerebroventricle as previously described (5). After a return to preimplantation body weight, an indwelling jugular vein cannula was implanted as previously described (17) under isoflurane-induced anesthesia (IsoSol, Vedco). The jugular cannula was exteriorized at the back of the neck and sealed with heparinized saline (200 U/ml 0.9% NaCl). On the following day, an extension line (PE-50) was attached to the jugular cannula to facilitate blood sampling, and the rats were left undisturbed for 1 h. An initial blood sample was removed from the jugular vein without disturbing the animal. All blood samples (0.3 ml) were removed from conscious, unrestrained rats into heparinized syringes and replaced with an equal volume of 0.9% NaCl (37 C). Blood samples were stored on ice before plasma was separated (10,000 x g for 5 min) and stored at –20 C until hormone assays (prolactin, GH, and corticosterone) were conducted. Immediately after the removal of the initial (0 time) blood sample, rats were administered 2 µl saline vehicle or saline containing 128, 256, or 768 pmol rat AM (Phoenix Pharmaceuticals, Belmont, CA) via the indwelling cerebroventricular cannula. Subsequent blood samples were removed 5, 15, 30, and 60 min after icv injections.
In another series of experiments, separate groups of rats were pretreated iv with saline vehicle or vehicle containing 0.2 mg CRH antagonist (
-helical CRF9–41, Phoenix Pharmaceuticals) immediately after the removal of the initial (0 time) blood sample. Fifteen minutes later a second blood sample was collected, and then animals received a central (icv) injection of 256 pmol AM in 2 µl saline or vehicle alone. Blood samples were collected as before 15, 30, 60, and 75 min after the icv injections.
RIAs
Plasma corticosterone levels were determined according to the instructions of the commercial RIA kit (rat/mouse corticosterone, ICN Biomedicals, Inc., Costa Mesa, CA). The minimum detectable hormone level was 25 ng/ml, and the inter- and intraassay coefficients of variability were less than 10%. Prolactin levels in plasma were determined using the kit materials provided by the National Hormone and Pituitary Program (rPRL-RP-3 standard). The minimum detectable hormone level in medium and plasma for prolactin was 0.5 ng/ml (defined as <90% B/B0) and the inter- and intraassay coefficients of variability were less than 9%. GH levels were similarly measured using the material provided in the National Institutes of Health (NIH) kit (rGH-RP-2 standard, minimum detectable level, 0.5 ng/ml; inter- and intraassay coefficients of variability were <8%).
Statistical analyses
Data were analyzed by ANOVA with Scheffé’s multiple comparison post hoc testing both within treatment groups across time and across treatment groups at any sampling time point. Results were considered significant if the probability was less than 5% (P < 0.05).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Saline vehicle injection did not significantly alter plasma corticosterone levels at any time point (within-group ANOVA). However, dose-related elevations in plasma corticosterone levels were observed after icv injections of AM (Fig. 1). Plasma corticosterone levels rose a maximum of 7.0-fold in the 128-pmol AM treatment group (fold change from baseline, within-group ANOVA, P < 0.001), 8.5-fold in the 256-pmol treatment group (P < 0.05) and 3.2-fold in the 768-pmol treatment group (P < 0.05). Plasma corticosterone levels began to rise 5 min after injection of 128 pmol AM and attained significance at 15 min (P < 0.01) and 30 min (P < 0.05 vs. time 0, within-group analysis). Elevations of plasma corticosterone levels after icv administration of 256 and 768 pmol AM were significant at 30 min (P < 0.01 vs. saline, between-group analyses; 256 pmol AM, P < 0.05, within-group analysis). By 60 min, plasma corticosterone levels in the 128- and 256-pmol AM-treated groups fell to levels that did not differ significantly from those present in saline vehicle-injected controls; however, the corticosterone levels remained elevated in animals receiving 768 pmol AM (P < 0.05, between-group ANOVA).
|
). In rats pretreated iv with saline vehicle, central administration of 256 pmol AM resulted in a significant elevation in plasma corticosterone levels (P < 0.001, within-group ANOVA). The iv administration of the CRH antagonist alone did not significantly alter plasma corticosterone levels at any time during the experiment (P = 0.213, within-group ANOVA). However, CRH antagonism significantly blunted the central AM-induced corticosterone rise (area under the curve for saline/saline was 543 ± 15 ng/ml·min; for CRH antagonist/saline, 357 ± 30 ng/ml·min; for saline/AM, 1115 ± 130 ng/ml·min; and for CRH antagonist/AM, 675 ± 74 ng/ml·min; P < 0.001). The only significant rise in plasma corticosterone levels in the CRH antagonist/AM-treated group was observed 30 min after central AM administration (P < 0.05, within-group ANOVA). Significantly lower corticosterone levels were observed between the CRH antagonist/AM-treated rats compared with saline/AM-treated rats at 15, 60, and 75 min (P < 0.05, between-group ANOVA, Scheffé’s multiple comparison testing).
|
). When compared with prolactin levels present in controls, a significant elevation was observed in the 256-pmol AM group at 15 and 30 min (P < 0.05, between-group ANOVA). Administration of the CRH antagonist alone did not alter plasma prolactin levels (P = 0.409) nor did it alter the prolactin rise induced by icv administration of AM (Fig. 3B). Plasma GH levels were not significantly altered by icv administration of saline vehicle or any dose of AM (Fig. 4).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The role of AM in the modulation of HPA axis activity appears to be quite complex. AM has been reported to have distinct actions at each level of the HPA axis. Data from studies conducted in rats (9) and sheep (8) indicated that central administration of AM elevated plasma ACTH levels. Our data (Fig. 1
) extend these findings by demonstrating that plasma corticosterone levels also rise in rats after icv administration of AM.
We previously have established that plasma corticosterone levels mirror circulating ACTH levels in nonstressed animals (18) and prefer this method because it requires only small aliquots of plasma and measures the business end of the HPA axis, the hormone that exerts the stress-related effects. For similar reasons, the measurement of plasma, urinary, or salivary cortisol levels is used in the clinical setting to assess HPA axis function. Although plasma corticosterone levels normally track changes in plasma ACTH concentrations, the half-lives of the two hormones differ and there is a time lag before elevated pulses of ACTH result in glucocorticoid secretion. Thus, it is possible, if very frequent sampling is not conducted, that changes in corticosterone levels may not truly mirror pituitary hormone release. Additionally, factors other than ACTH can affect corticosterone secretion (e.g. sympathetic nerve activation and circulating vasoactive hormones), and as a result, increases or decreases in plasma glucocorticoid levels may not solely be a result of changes in ACTH secretion. Because we are studying the effect of a peptide that when administered icv elevates sympathetic tone (2, 3) in addition to apparently stimulating the release of CRH (8, 9) (Fig. 2), we feel that in the end, corticosterone measurements give us the best window through which to examine the endpoint of HPA axis activation.
Our data demonstrating that icv administration of AM elevated plasma corticosterone levels (Fig. 1) suggest that AM acts within the hypothalamus to stimulate CRH release into the hypophyseal portal vessels, thus increasing activity of the HPA axis. Additional data that predicted that central administration of AM may lead to CRH release include the following: AM has been shown to induce Fos expression in CRH-positive hypothalamic PVN neurons (9), and AM directly caused depolarization of parvocellular PVN neurons in brain slices (10).
Neurons in the parvocellular PVN have been segregated into at least two classes: neuroendocrine, projecting to median eminence, and preautonomic, those projecting to cardiovascular centers in brain stem (11, 12, 13). Of the neuroendocrine cells in parvocellular PVN, some produce CRH, whereas others express the genes for TRH, GHRH, somatostatin, or tyrosine hydroxylase (14). Thus, the depolarizing effect of AM on parvocellular neurons in PVN (10) may have reflected an action of endogenous peptide to activate preautonomic projection neurons, to activate neuroendocrine cells resulting in the stimulation of ACTH, PRL, or GH release, or on the other hand to inhibit the release of PRL or GH. Furthermore, the CRH-positive neurons in PVN affected by AM administration (9) may not in fact be neuroendocrine neurons but instead CRH-positive elements that relay autonomic or behavioral information (15, 16). Indeed, CRH expression and secretion has been shown to be differentially regulated in these functionally distinct neurons (15, 16). Therefore, although early results suggested that central AM may activate the HPA axis through the release of CRH, a conclusive study has not been previously conducted. Using a CRH antagonist we were able to abrogate the ability of central AM to stimulate the HPA axis (Fig. 2), supporting the hypothesis that AM activates the HPA axis, at least in part, by an action in the hypothalamus that results in increased CRH release into the portal vessels.
Although CRH is thought to be the major modulator of axis activity, several other neural and hormonal pathways have been demonstrated to augment the CRH-induced activation of the HPA axis under various circumstances; among these factors are vasopressin (AVP), oxytocin (OT), angiotensin II, and catecholamines (19, 20). Furthermore, glucocorticoids, the end product of HPA activation, feed back to inhibit CRH-induced ACTH release (19). Although CRH antagonism significantly reduced the AM-induced corticosterone rise (Fig. 2), the failure of the CRH antagonist to completely block the AM-induced corticosterone rise could be due to elevated portal blood levels of AVP, OT, or another hormone (20) or to descending sympathetic activity via the splanchnic nerve (21).
The potential contribution of AVP, OT, and the sympathetic nervous system to the AM-induced activation of the HPA axis could be established using additional experimental approaches. Plasma corticosterone levels could be measured after administration of an AVP or OT antagonist by itself or in combination with the CRH antagonist (20) to determine whether these antagonists could further dampen the corticosterone response to central AM. Alternatively, plasma or, better yet, portal blood AVP and OT levels could be measured in a similar experimental paradigm using only the CRH antagonist; however, large quantities of plasma are needed to measure these hormones (AVP, 1 ml; OT, 0.3 ml) and, therefore, would require collection of trunk blood from rats and permit sampling at only a single time point in each rat. In the studies described here, to avoid hypovolemic challenge, we collected only 0.3 ml whole blood (
0.15 ml plasma) at each sampling, allowing us to measure corticosterone, prolactin, and GH at multiple time points in the same rat, but insufficient plasma for accurate determination of AVP and OT levels.
It is possible that icv administration of AM could increase splanchnic nerve activity because the peptide has been demonstrated to act in brain to increase sympathetic tone (2, 3). To determine the contribution of the sympathetic nervous system to the AM-induced corticosterone rise, experiments could be performed in rats whose splanchnic nerves had been cut, or plasma ACTH levels could be measured in a repeat of the CRH antagonist studies. Again, however, larger quantities (0.1–1.0 ml) of plasma are needed to assess ACTH levels than were collected in our studies. Nevertheless, the AM-induced rise in plasma corticosterone levels seen in CRH antagonist-pretreated rats was not of the magnitude of that seen in saline vehicle-pretreated rats (Fig. 2), suggesting that a major effect of AM given icv is on CRH release.
It is clear from our data that icv administration of AM activates the HPA axis; however, the physiological role of central AM during stress remains unknown at this time. Mimoto and colleagues (22) reported that icv administration of AM did not alter plasma ACTH, corticosterone, or OT responses to severe physical stress. The regulation of the HPA axis by central AM during other forms of stress (minor physical, anticipatory, or social stresses) has not yet been examined. Indeed, differential modulation of CRH, AVP, and OT release by various stressors has been demonstrated (23, 24), suggesting that central AM may play a physiological role in coordinating the hormonal response to other forms of stress. Here, our studies were completed in unstressed animals and, therefore, focus on basal corticosterone levels as a mirror of tonic, overall HPA activity.
Our data, in conjunction with previous studies (9, 10), clearly demonstrate a role for AM in the hypothalamic branch of the HPA axis. The roles of AM in the pituitary and adrenal glands, however, remain controversial. When given iv, where the peptide can have effects at the level of either the pituitary or adrenal gland, AM has been reported to cause disparate changes in the HPA axis depending on species. In humans, iv infusion of AM did not alter basal ACTH (25) or cortisol (25, 26) levels. However, AM was able to augment ACTH-stimulated cortisol secretion in humans (26). This may have been secondary to the hypotensive action of AM given iv and not a reflection of a direct effect of the peptide on the adrenal or pituitary glands. On the other hand, in sheep, iv infusion of AM reduced circulating ACTH and cortisol levels by more than 50% (27). Although there are no reports of the effects of iv administration of AM on ACTH or corticosterone in rats, tissue-specific effects have been examined. Our group has previously reported that AM inhibits both basal and CRH-stimulated ACTH release from dispersed anterior pituitary glands harvested from male rats (28). Mimoto and colleagues (22) reported that AM did not alter basal or CRH-stimulated ACTH release from dispersed anterior pituitary cells but did inhibit OT-stimulated ACTH release. The effects of AM on corticosterone release from dispersed adrenal cells are similarly unclear. One group reported that AM had no effect on basal or ACTH-stimulated corticosterone release from dispersed adrenocortical cells (29), although another group reported that AM did not alter basal corticosterone secretion but stimulated ACTH-induced corticosterone secretion (30). Nevertheless, the finding that AM might stimulate the HPA axis within the hypothalamus while inhibiting it at another level is not without precedent as this phenomenon also has been demonstrated with other peptides (18, 31).
The stress response in rats, as well as humans, not only comprises changes in the HPA axis but also includes alterations in the secretion of other hormones. We examined the potential modulation of the secretions of two additional stress hormones, prolactin and GH, by central AM. Plasma prolactin levels were elevated by icv administration of AM (Fig. 3), a response similar to that observed after icv administration of other neuropeptides (32). The central AM-induced elevation in prolactin might be due to the withdrawal of inhibitory hypothalamic dopamine tone as is seen with other peptides (32). Recent reports, however, indicate that AM may stimulate neuronal dopamine systems to cause diuresis and natriuresis (33). It may be possible that AM stimulates dopamine release in one neuronal circuit in brain and inhibits dopamine turnover in another or that AM stimulates the release of a yet to be identified prolactin-releasing factor (34). Unlike other potent stress peptides (32, 35), central AM had no effect on GH levels (Fig. 4). Thus, the actions of AM in brain may reflect the peptide’s selective effect in the PVN, particularly on the parvocellular division. Although it is now clear that AM can activate CRH neurons, the peptide has been reported to hyperpolarize magnocellular PVN neurons in brain slices (10), suggesting that OT and AVP likely do not play a role in the AM-induced activation of the HPA axis.
In conclusion, the findings reported here lead us to propose that AM may be an important neuromodulator of the hormonal stress response. They add to our growing hypothesis about the role of AM in the physiological response to stress and suggest site-specific actions of AM that activate the HPA axis at the hypothalamic level and inhibit the HPA axis at the pituitary level, assuring, therefore, the animal’s hormonal response to stress is appropriate, but not excessive. Clearly, studies examining stress hormone secretion during blocked action or decreased production of brain-derived AM are needed to confirm the role of central AM in the physiological response to stress.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: AM, Adrenomedullin; AVP, vasopressin; HPA, hypothalamic-pituitary-adrenal; icv, intracerebroventricular; OT, oxytocin; PVN, paraventricular.
Received June 28, 2004.
Accepted for publication July 16, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  E. Marinoni, C. Zacharopoulou, A. Di Rocco, C. Letizia, M. Moscarini, and R. Di Iorio Effect of Betamethasone In Vivo on Placental Adrenomedullin in Human Pregnancy Reproductive Sciences, September 1, 2006; 13(6): 418 - 424. [Abstract] [PDF]
|
|
|
|
 |
|
 M. M. Taylor, S. L. Bagley, and W. K. Samson Intermedin/Adrenomedullin-2 Inhibits Growth Hormone Release from Cultured, Primary Anterior Pituitary Cells Endocrinology, February 1, 2006; 147(2): 859 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lin Chang, J. Roh, J.-I. Park, C. Klein, N. Cushman, R. V. Haberberger, and S. Y. T. Hsu Intermedin Functions as a Pituitary Paracrine Factor Regulating Prolactin Release Mol. Endocrinol., November 1, 2005; 19(11): 2824 - 2838. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
