Ouabain has been identified in the plasma and adrenal glands of several mammals, including humans. To investigate possible adrenal secretion of ouabain in vivo, at rest, and in response to acute blood volume changes, we prepared trained adult dogs (n = 10) with splenectomy and unilateral adrenal venous (AV) cannulation. Two days later, after an overnight fast, dogs had either 1) 20% hemorrhage (hem) or 2) 20% blood volume expansion (exp; 6% Dextran 70, 0.9% NaCl) in random order. In AV and arterial plasma (ART), ouabain was measured by a ouabain-specific immunoassay, and cortisol and aldosterone were measured by radioimmunoassay. ART and AV ouabain concentration did not change after hem or exp [P = not significant (NS)]. In 94 of 97 paired samples, the concentration of ouabain in AV was greater than that in ART (Wilcoxon, P < 0.001), and the mean ouabain concentration was greater in AV (756.4 +/- 85.7 pmol/l) than ART (235.4 +/- 18.5 pmol/l; P < 0.001). The mean AV-to-ART ouabain concentration ratio was 5.7 +/- 1.29. Adrenal secretion of ouabain was not influenced by hem or exp (analysis of variance, P = NS). Adrenal secretion of cortisol and aldosterone increased after hem (P < 0.05) and was unaltered by exp (P = NS). This study demonstrates that ouabain is secreted by the adrenal gland in the awake dog. However, adrenal ouabain secretion and arterial blood ouabain are not altered by acute hem or exp.

We studied the effect of surgery on hemorrhage-induced facilitation in the pituitary-adrenal system using repeated hemorrhage in chronically prepared dogs. Animals underwent splenectomy and adrenal venous and femoral arterial catheterization. Two (day 2) or five (day 5) days later, animals were anesthetized with pentobarbital, respired, and subjected to two periods of hypovolemia (20% hemorrhage with reinfusion of shed blood at 30 min; H1 and H2) separated by 90 min. Arterial and adrenal venous blood was sampled, and adrenocorticotropic hormone (ACTH), arginine vasopressin (AVP), angiotensin II (ANG II), cortisol, and cortisol secretion were measured. On day 2, cortisol secretion increased similarly after H1 and H2, despite a smaller response of ACTH to H2. On day 5, neither ACTH nor cortisol secretion changed after H1, but both increased significantly after H2. The adrenal sensitivity to ACTH increased after H2 on day 2 and was similar after H2 on both days. AVP and ANG II increased similarly after H1 and H2 on each day but had larger responses on day 2. These results suggest 1) either surgery or initial hemorrhage can lead to enhanced pituitary-adrenal responses to subsequent hemorrhage, 2) this effect may have both central and adrenal components, 3) negative feedback may inhibit enhanced responses of ACTH in this model, 4) changes in adrenal sensitivity to ACTH may not depend on an initial pituitary-adrenal response and may not be blocked by increased circulating corticosteroids, and 5) differences in circulating AVP or ANG II do not account for facilitation in pituitary-adrenal responses.

Potentiated pituitary-adrenocortical responses to the second of two identical small hemorrhages, spaced 24 h apart, are seen in the pentobarbital sodium-anesthetized dog. To investigate the role of pentobarbital anesthesia in these results and to better define the range of the effect, we studied awake trained dogs with chronic adrenal venous catheters. Each dog was bled an amount between 8.7 and 21.8% of measured blood volume [131I] (MBV) over 3 min, and peripheral and adrenal blood were sampled. Blood was reinfused 1 h later, and the dogs were fed. The same hemorrhage and experimental protocol were repeated 24 h later. Steroids were assayed by high-performance liquid chromatography-ultraviolet (HPLC-UV) and adrenocorticotropic hormone (ACTH) by radioimmunoassay (RIA). Secretory rates of cortisol were calculated using measured adrenal blood flow rates. Maximal secretion of cortisol was determined after injection of 100 mU ACTH following each experiment. Dogs whose day 1 cortisol secretion after hemorrhage was submaximal (hem volume = 14.8 +/- 3.7% MBV; n = 7) showed a greater cortisol secretory response to the same hemorrhage on day 2 (P less than 0.005). This increased cortisol response on day 2 was accompanied by an increased ACTH presentation rate (APR) (P less than 0.025) and by increased adrenal sensitivity to ACTH (P less than 0.025). The increased APR was caused by both an increased venous ACTH and by an increased adrenal blood flow. If posthemorrhage cortisol secretion was maximal on day 1, ACTH, APR, and ABF were not different on the 2 days. No hemodynamic differences were seen to explain these findings. These results confirm and extend our previous results.(ABSTRACT TRUNCATED AT 250 WORDS)

We determined how changes in the responsiveness of the hypothalamo-pituitary-adrenal (HPA) system that accompany experimentation affect facilitation of HPA responses to hemorrhage. Hemorrhage (10 ml/kg over 3 min) was performed in conscious, chronically prepared rats. Blood was sampled over 1 h followed by reinfusion of shed blood. Hemorrhage was performed either once or twice separated by 24 h in different groups of animals. To test the effect of the circadian variation in responsiveness, rats were hemorrhaged on days 4 and 5 after surgery either in the morning (AM) or in the afternoon (PM). The response of ACTH to hemorrhage on day 4 was greater in the PM than in the AM (P<0·01). The ACTH response to the second hemorrhage on day 5 was greater than that to hemorrhage on day 4 only in the AM group (P<0·01). Thus, facilitation of ACTH responses by prior hemorrhage was evident only in the AM. To determine the effects of surgical recovery, additional experiments were done in the AM either early (days 3 and 4) or later (days 6 and 7) after surgery. In these experiments, hemorrhage was performed in all rats on days 4 and 7 and either hemorrhage or blood sampling alone was performed on day 3 and 6. ACTH did not increase in rats with sampling and no hemorrhage. ACTH increased more after an initial hemorrhage on day 3 than on day 6 (P<0·01). ACTH response to hemorrhage on day 4 was greater when preceded by hemorrhage vs sampling on day 3 (P<0·01). ACTH response to hemorrhage in rats bled twice did not differ on day 3 and day 4. On day 7, the response of ACTH in rats that had hemorrhage on day 6 was greater than both their own response on day 6 and the response of a control group with sampling on day 6 (P<0·01). These results demonstrate potentiation of ACTH responses to hemorrhage by an earlier similar hemorrhage, but clearly indicate that enhanced sensitivity of the HPA to hemorrhage either by circadian factors or by surgery can mask this effect.

To test whether corticotropin-releasing factor (CRF) is a secretagogue for adrenal secretion of catecholamines, a preparation was developed that permits measurement of adrenal venous output in response to in vivo arterial injection into the dog adrenal gland. Dogs were prepared with catheters in the lumboadrenal vein for monitoring adrenal blood flow and secretion rate of epinephrine and norepinephrine and in the lumboadrenal and inferior phrenic arteries for adrenal injection. Under pentobarbital anesthesia 48 h after surgery, dogs received a series of intra-adrenal injections that included acetylcholine (0.2-200 nmol), CRF (2.0-20.0 nmol), and diluent. There was a log dose-response relationship before epinephrine secretion and norepinephrine secretion to acetylcholine. Adrenal injection of CRF stimulated epinephrine and norepinephrine secretion at the highest dose tested (20 nmol). The response observed was equivalent to the response to 0.2 nmol acetylcholine. A similar dose of CRF given systemically produced hypotension without stimulating catecholamine responses. Adrenal catecholamine responses to acetylcholine were not augmented by addition of CRF. These findings show that arterial injection of CRF into the intact dog adrenal stimulates secretion of epinephrine and norepinephrine. However, the low potency of CRF relative to that of acetylcholine, the lack of a synergistic effect of CRF on catecholamine responses to acetylcholine, and the high dose of CRF required to achieve a response suggest that CRF does not function in the adrenal medulla as a physiologically important secretagogue for catecholamines.