removal of 15% of the rat's estimated blood volume (7.25% of body weight) at the rate of 5%/min from an arterial cannula. Shed blood was withheld from the ...
0013-7227/85/1162-0633$02.00/0 Endocrinology Copyright© 1985 by The Endocrine Society
Vol. 116, No. 2 Printed in U.S.A.
Evidence for Multifactor Regulation of the Adrenocorticotropin Secretory Response to Hemodynamic Stimuli* PAUL M. PLOTSKY, THOMAS 0. BRUHN, AND WYLIE VALEf Peptide Biology Laboratory, The Salk Institute, La Jolla, California 92037
ABSTRACT. We have examined the contributions of corticotropin-releasing factor (CRF), arginine vasopressin (AVP), epinephrine, and oxytocin to the ACTH secretory responses to hemorrhage. The relative significance of each of these putative ACTH regulatory factors is undefined with respect to net ACTH secretion. Initially, the effects of selective systemic pharmacological blockade of individual factors on the ACTH response were examined. Immunoneutralization of CRF reduced resting ACTH levels below the detection limits of our RIA and abolished the secretory response to hemorrhage. Ganglionic blockade or treatment with a potent AVP antagonist reduced the ACTH secretory response by 55% and 38%, respectively. Further evidence for multifactor regulation of hemodynamically evoked alterations in ACTH secretion was obtained by measurement of
the concentrations of these factors in the hypophysial portal circulation during hemorrhage. Immunoreactive CRF, AVP, oxytocin and epinephrine were present in the portal plasma at concentrations within a range shown to evoke ACTH secretion from cultured pituitary cells when presented alone or in combination. The concentrations of all of these were significantly elevated during hemorrhage. During atrial pulsation, a stimulus mimicking volume loading and associated with a reduction of systemic ACTH levels, we observed a significant decline in portal concentrations of immunoreactive AVP coupled with a nonsignificant trend toward reduced portal immunoreactive CRF levels. These observations are highly suggestive of multifactor regulatory control of ACTH secretion in response to hemodynamic stimuli. (Endocrinology 116: 633-639, 1985)
14) and oxytocin (OT)1 (11) have ACTH-releasing activity either alone or in combination with CRF in vitro. Furthermore, the presence of all of these putative regulatory factors in the hypophysial portal circulation of the rat has been reported (3,15-19). The purpose of these studies was to test the hypothesis of multifactor regulation of ACTH secretion in response to hemodynamic stimuli. A pharmacological approach was followed to characterize the contributions of CRF, AVP, and E to hemorrhage-induced ACTH secretion. Hypophysial portal plasma levels of E, irCRF, irAVP, and irOT were also measured during hemorrhage and simulated volume loading in an attempt to correlate hypothalamic hypophysial and anterior pituitary secretory events.
ADENOHYPOPHYSIAL secretion of ACTH is regu1 1 lated in a complex manner which may involve multiple regulatory factors of hypothalamic, neurohypophysial, and peripheral origins (1, 2). We have previously demonstrated the dynamic role of immunoreactive corticotropin-releasing factor (irCRF) in mediation of hemorrhage-induced ACTH secretion (3) and alluded to the potential involvement of additional regulatory factors. Support for this concept may be derived from the observations of Rivier et al. (4, 5) and Bruhn et. al. (6) with respect to attenuation of ether stress-induced ACTH secretion by selected pharmacological interventions. High affinity receptors for CRF (7), arginine vasopressin (AVP) (8), and epinephrine (E) (9, 10) have been identified in adenohypophysial tissue. These agents (11-
Materials and Methods
Received May 29,1984. Address requests for reprints to: Dr. Paul M. Plotsky, Peptide Biology Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. * This work was supported by NIH Grant AM-33093 (to P.M.P.), the Mellon Foundation (to P.M.P.), Deutsche Forschungsgemeinschaft Grant BR-794/1-1 (to T.O.B.), and the Clayton Foundation for Research, California Division. Presented in part at the Kroc Foundation Conference on Corticotropin-Releasing Factor, Solvang, CA, February 20-24, 1984. t Investigator, Clayton Foundation.
Animals Experiments were performed on urethane-anesthetized male rats of the Charles River-derived Sprague-Dawley strain (325375 g). Rats were maintained in environmentally controlled quarters on a 12-h light, 12-h dark cycle, with food and water available ad libitum. Contemporaneous experiments were carried out in separate groups of rats to characterize the ACTH 633
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REGULATION OF ACTH RESPONSE TO HEMODYNAMIC STIMULI
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and hypophysiotropic secretory responses to the stimuli presented. Surgical procedures Surgical preparation of the animals was performed in the morning, while experimental data collection was done from 1300-1700 h. Animals were treated with 2% xylocaine at all points of pressure or incision in an attempt to minimize pain. Body temperature was maintained at 37 ± 1 C. An endotracheal tube was placed to insure patency of the airway, and polyethylene cannulae (id, 0.58 mm; od, 0.97 mm) were placed in the descending aorta via the femoral arteries for systemic sampling and monitoring of cardiovascular parameters. A femoral venous line was placed for fluid support (lactated Ringer's solution) and drug administration. Sequential collections of hypophysial portal blood were obtained from the transected infundibular stalk by a modification (20) of the parapharyngeal approach originally described by Porter (21). Blood sampling Sampling commenced after a 60-min postsurgical stabilization period. Peripheral samples (0.5 ml) were collected at multiple time points for measurement of ACTH and E levels during the hemorrhage and atrial pulsation paradigms. Sample volume was replaced by infusion of heparinized (10 IU/ml) normal saline. Hypophysial portal samples were obtained in sequential collections at the rate of 6-10 jil/min via a syringe pump (Harvard Apparatus, Millis, MA). Peripheral samples were withdrawn immediately before and 30 min after stalk transection for assessment of systemic levels of ACTH, irCRF, irAVP, and irOT. Blood was kept refrigerated until centrifugation, and plasma was stored at —20 C until assay. Hormone measurements The RIA for ACTH was performed, as previously described (22), using antiserum provided by Dr. David Orth. The detection limit of the assay was 2-4 pg/tube, and the intra- and interassay coefficients of variance were 6% and 10%, respectively. E was extracted from plasma with acid-activated alumina and subsequently quantified using HPLC coupled with electrochemical detection. The detection limit of the assay was 15 pg, with a 3% intraassay and a 7% interassay coefficient of variance. A complete description of the technique was recently presented (23). CRF, AVP, and OT were measured by RIA on samples extracted using Bond-Elut C18 columns (Analytichem, Harbor City, CA). CRF was eluted with 2-propanol with a recovery of 70% or more. The neurohypophysial peptides were eluted from C18 columns with an acetonitrile-triethylammoniumformate mixture (75%:25%), with a recovery of more than 90%. Sample elutants were evaporated to dryness and redissolved in assay buffer. Each sample was split 70% for CRF and 15% each for AVP and OT assays. Rabbit antiserum rC-68 to synthetic rat CRF was used in a final dilution of 1:1,000,000 for the CRF RIA, as recently described (24). The response of portal plasma sample dilutions was parallel to that of plasma containing synthetic CRF, AVP, and OT, as shown in Fig. 1. RIAs of AVP and OT were performed according to the
O
1
10 pg OT
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FIG. 1. A, CRF standard curve and parallelism of dilutions of hypophysial portal plasma extracts. The limit of detection and ICso of this assay were 2 and 22 pg, respectively. The intraassay coefficient of variation was 3.6%, while the interassay coefficient of variation was 10.6%. B, AVP standard curve and parallelism of dilutions of rat hypophysial portal plasma extracts. The minimum amount of AVP that could be detected was 0.7 pg, and the ICM was 10 pg. Intra- and interassay coefficients of variation were 6.5% and 11.7%, respectively. C, OT standard curve and dilutions of portal plasma extracts. The limit of detection and IC50 were 0.5 and 8 pg, respectively. Coefficients of variation were similar to those of the AVP assay. B/BO, Bound to free ratio; A- -A systemic plasma; O- -O, hypophysial portal plasma; • — # , synthetic standard.
procedure of Weitzman et al. (25) using rabbit antiserum to both peptides prepared by Dr. D.*A. Fisher. AVP antiserum R71 was used at a final dilution of 1:112,000 and exhibited crossreactivity of less than 0.02% with synthetic OT. OT antiserum R-421 was used at a final dilution of 1:12,800 and exhibited 0.2% cross-reactivity with synthetic AVP. Experimental treatments Hemorrhage, which has been demonstrated to reliably evoke increased ACTH secretion in anesthetized rats (27), involved
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REGULATION OF ACTH RESPONSE TO HEMODYNAMIC STIMULI removal of 15% of the rat's estimated blood volume (7.25% of body weight) at the rate of 5%/min from an arterial cannula. Shed blood was withheld from the circulation for 27 min in a warmed heparinized syringe, then reinfused at 5%/min. Pharmacological treatments before systemic blood collection protocols included 1) vehicle injection; 2) injection 10 min before hemorrhage of deaminopenicillamine-2-(O-methyl)tyrosine vasopressin (150 fig/kg BW, iv), a potent antagonist of the pressor (26) and ACTH-releasing (28) activities of AVP; 3) injection 60 min before hemorrhage of chlorisondamine (3 mg/ kg BW, ip), a peripheral ganglionic blocking agent; or 4) immunoneutralization 10 min before hemorrhage of CRF by injection of rabbit antiserum to synthetic rat CRF (0-1.0 ml rC70, iv). Atrial pulsation has been used to simulate volume loading in the anesthetized cat by Gann et al. (29, 30). They demonstrated decreased ACTH levels in the peripheral circulation in response to direct activation of right atrial and septal jS-receptors with atrial pulsation. We have modified their protocol for use in the rat to provide a controlled stimulus, resulting in decreased ACTH secretion. A vinyl cannula filled with heparinized saline (25 IU/ml) was advanced into the right atrium from the jugular vein. A volume of 0.20 ml was infused and withdrawn sinusoidally for 30 min at a rate of 2 Hz via a modified syringe pump.
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• VEHICLE (n-6)
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FlG. 2. ACTH secretion in response to hemorrhage; attenuation after administration of pharmacological agents. Urethane-anesthetized male rats were hemorrhaged by withdrawal of 15% of the estimated blood volume from an arterial cannula at a rate of 5%/min. Blood was withheld for 27 min, then reinfused. Systemic ACTH concentrations in vehicle-treated rats were significantly elevated us. initial levels at +5 min (P < 0.05) and +10, 20, 30, and 45 min (P < 0.01). Drug treatment significantly attenuated this response (see Results). HEM, Hemorrhage; R, reinfusion.
Statistical analyses
o 1.00 cc NRS (n-5)
Raw data were analyzed by single or multifactor analysis of variance techniques for significant overall effects. These effects were evaluated using Duncan's new multiple range test (31). A P value less than 0.05 was regarded as significant. Data are presented as the mean ± SEM.
Results Hemorrhage-induced ACTH secretion Hemorrhage evoked a maximal 4-fold increase in the systemic ACTH concentration by 10 min (n = 6) from a prehemorrhage mean concentration of 183 ± 46 pg/ml (Fig. 2, vehicle). Systemic ACTH remained significantly elevated (P < 0.01) throughout the period of hypovolemia and for 30 min after reinfusion. While prehemorrhage ACTH levels were unaffected by systemic pretreatment with an AVP antagonist (n = 6) or a ganglionic blocking agent (n = 6), the secretory response to hemorrhage was attenuated with both agents. Peak ACTH levels were reduced by 37.8 ± 6.5% (P < 0.01) from an initial mean level of 212 ± 42 pg/ml after AVP antagonist treatment. Interestingly, the early phase (+5 min) of the response to hemorrhage appeared to be facilitated by AVP antagonist treatment (P < 0.05 vs. vehicle). The ACTH levels in this group were significantly lower than those attained in the vehicle group during (+10, 20, and 30) and immediately after (+45) hemorrhage (P < 0.01). Chlorisondamine treatment attenuated the peak ACTH secretory response to hemorrhage by 55.2 ± 5.7% (P < 0.01). The cumulative secre-
• 0.10 cc abCRF (n-6) 800-
A 0.26 cc abCRF (n-6) • 0.6 cc abCRF (n-6)
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