Adrenal Venous Plasma Research Articles

Cholesterol delivery to the adrenal glands estimated by adrenal venous sampling: An in vivo model to determine the contribution of circulating lipoproteins to steroidogenesis in humans

Cholesterol, required for adrenal steroid hormone synthesis, is at least in part derived from circulating lipoproteins. The contribution of high-density lipoproteins (HDL) and low-density lipoproteins (LDL) to adrenal steroidogenesis in humans is unclear. The aim of the study was to determine the extent to which HDL and LDL are taken up by the adrenal glands using samples obtained during adrenal venous sampling (AVS). AVS was successfully performed in 23 patients with primary aldosteronism. Samples were drawn from both adrenal veins and inferior vena cava (IVC). HDL cholesterol (HDL-C) and lipoprotein particle profiles were determined by nuclear magnetic resonance spectroscopy. Apolipoprotein (apo) A-I and apoB were assayed by immunoturbidimetry. Plasma HDL-C and HDL and LDL particle concentrations (HDL-P and LDL-P) were not lower in samples obtained from the adrenal veins compared with the IVC (HDL-C, P=.59; HDL-P, P=.06; LDL-P, P=.93). ApoB was lower in adrenal venous plasma than in IVC (P=.026; P<.05 for right adrenal vein). In 13 patients with an aldosterone producing adenoma (APA), apoB was also lower (P=.045) and LDL-P tended to be lower (P=.065) in the APA adrenal vein compared with the IVC. ApoA-I was not lower in adrenal venous plasma compared with the IVC, neither in the whole group (P=.20) nor in the APA subgroup (P=.075). These invivo observations suggest that circulating LDL may contribute to adrenal steroidogenesis in humans as inferred from adrenal venous-IVC apoB concentration differences. AVS is a feasible method to investigate the relationships between lipoproteins and steroidogenesis.

Mass Spectrometry-Based Adrenal and Peripheral Venous Steroid Profiling for Subtyping Primary Aldosteronism.

Differentiating patients with primary aldosteronism caused by aldosterone-producing adenomas (APAs) from those with bilateral adrenal hyperplasia (BAH), which is essential for choice of therapeutic intervention, relies on adrenal venous sampling (AVS)-based measurements of aldosterone and cortisol. We assessed the utility of LC-MS/MS-based steroid profiling to stratify patients with primary aldosteronism. Fifteen adrenal steroids were measured by LC-MS/MS in peripheral and adrenal venous plasma from AVS studies for 216 patients with primary aldosteronism at 3 tertiary referral centers. Ninety patients were diagnosed with BAH and 126 with APAs on the basis of immunoassay-derived adrenal venous aldosterone lateralization ratios. Among 119 patients confirmed to have APAs at follow-up, LC-MS/MS-derived lateralization ratios of aldosterone normalized to cortisol, dehydroepiandrosterone, and androstenedione were all higher (P < 0.0001) than immunoassay-derived ratios. The hybrid steroids, 18-oxocortisol and 18-hydroxycortisol, also showed lateralized secretion in 76% and 35% of patients with APAs. Adrenal venous concentrations of glucocorticoids and androgens were bilaterally higher in patients with BAH than in those with APAs. Consequently, peripheral plasma concentrations of 18-oxocortisol were 8.5-fold higher, whereas concentrations of cortisol, corticosterone, and dehydroepiandrosterone were lower in patients with APAs than in those with BAH. Correct classification of 80% of cases of APAs vs BAH was thereby possible by use of a combination of steroids in peripheral plasma. LC-MS/MS-based steroid profiling during AVS achieves higher aldosterone lateralization ratios in patients with APAs than immunoassay. LC-MS/MS also enables multiple measures for discriminating unilateral from bilateral aldosterone excess, with potential use of peripheral plasma for subtype classification.

Genotype-Specific Steroid Profiles Associated With Aldosterone-Producing Adenomas.

Primary aldosteronism comprises 2 main subtypes: unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia. Somatic KCNJ5 mutations are found in APA at a prevalence of around 40% that drive and sustain aldosterone excess. Somatic APA mutations have been described in other genes (CACNA1D, ATP1A1, and ATP2B3) albeit at a lower frequency. Our objective was to identify genotype-specific steroid profiles in adrenal venous (AV) and peripheral venous (PV) plasma in patients with APAs. We measured the concentrations of 15 steroids in AV and PV plasma samples by liquid chromatography-tandem mass spectrometry from 79 patients with confirmed unilateral primary aldosteronism. AV sampling lateralization ratios of steroids normalized either to cortisol or to DHEA+androstenedione were also calculated. The hybrid steroid 18-oxocortisol exhibited 18- and 16-fold higher concentrations in lateralized AV and PV plasma, respectively, from APA with KCNJ5 mutations compared with all other APA combined together (P<0.001). Lateralization ratios for the KCNJ5 group were also generally higher. Strikingly, we demonstrate that a distinct steroid signature can differentiate APA genotype in AV and PV plasma. Notably, a 7-steroid fingerprint in PV plasma correctly classified 92% of the APA according to genotype. Prospective studies are necessary to translate these findings into clinical practice and determine if steroid fingerprinting could be of value to select patients with primary aldosteronism who are particularly suitable candidates for adrenal venous sampling because of a high probability of having an APA.

An LC–MS/MS method for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism

BackgroundSteroid profiling for diagnosis of endocrine disorders featuring disordered production of steroid hormones is now possible from advances in liquid chromatography with tandem mass spectrometry (LC–MS/MS). Adrenal venous (AV) measurements of aldosterone and cortisol are a standard practice in the clinical work-up of primary aldosteronism, but do not yet take advantage of steroid profiling. MethodsA novel LC–MS/MS based method was developed for simultaneous measurement of 15 adrenal steroids: aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, pregnenolone, cortisone, cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, 21-deoxycortisol, 18-oxocortisol and 18-hydroxycortisol. These were compared in peripheral venous (pV) and AV plasma from 70 patients undergoing AV sampling with and without cosyntropin stimulation. Aldosterone and cortisol levels measured by LC–MS/MS were compared with those measured by immunoassay. ResultsReproducibility of measurements with coefficients of variation ≤10% as well as analytical sensitivity sufficient to measure low pV levels particularly of aldosterone demonstrate the utility of the assay for profiling adrenal steroids in primary aldosteronism. Method comparisons indicated assay and concentration dependent differences of cortisol and aldosterone concentrations measured by immunoassay and LC–MS/MS. Median AV/pV ratios of 11-deoxycortisol (53.0), 17-hydroxyprogesterone (33.4), pregnenolone (62.4), androstenedione (40.6) and dehydroepiandrosterone (33.3) were 2.9- to, 5.4-fold larger than those for cortisol (11.6), with additionally generally larger increases than for cortisol with than without cosyntropin stimulation. ConclusionOur LC–MS/MS assay, in addition to improvements over existing immunoassay measurements of aldosterone and cortisol, offers profiling of 13 other adrenal steroids, providing a potentially useful method for the clinical work-up of patients with primary aldosteronism. In particular, the larger AV/pV ratios of several steroids compared to cortisol suggest more sensitive alternatives to the latter for assessing positioning of AV sampling catheters.

Utility of LC-MS/MS based adrenal venous steroid profiling: Should cortisol remain the gold standard for selectivity and comparisons to aldosterone?

Background: Comparisons of aldosterone with cortisol during adrenal venous sampling (AVS) provide the currently recommended technique for distinguishing unilateral adenomas from bilateral adrenal hyperplasia during the diagnostic work-up of patients with primary aldosteronism. Selectivity of AVS is routinely established from adrenal venous (AV) to peripheral venous (PV) ratios of cortisol, but is more accurately indicated by AV: PV ratios of plasma metanephrine. Objective: Here we describe a novel liquid-chromatography tandem mass spectrometric (LC-MS/MS) assay for profiling plasma levels of 15 adrenal steroids during AVS, with and without ACTH stimulation. Methods: Selectivity of AVS was established by AV: PV ratios of plasma metanephrine larger than 12. Concentrations of aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, pregnenolone, cortisone, cortisol, 11-deoxycortisol, 17-hydroxy-progesterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, 21-deoxycortisol, 18-oxo-cortisol and 18-hydroxy-cortisol were compared in PV and AV plasma samples from 35 patients with and without ACTH stimulation. Aldosterone and cortisol levels measured by LC-MS/MS were compared with levels measured by immunoassay. Results: Median AV: PV ratios of plasma cortisol (17) were lower than those of corticosterone (52), 11-deoxycorticosterone (68), pregnenolone (72), 11-deoxycortisol (55) and 17-hydroxy-progesterone (57). ACTH stimulation increased AV plasma concentrations of cortisol 6-fold, but had larger effects on other steroids such as corticosterone, which increased by 36-fold. As a result AV plasma concentrations of corticosterone relative to cortisol increased from 6% to 38%. Method comparisons indicated agreement of concentrations of cortisol measured by immunoassay and LC-MS/MS. Conclusion: Compared to other adrenal steroids, cortisol provides a less sensitive marker for selectivity of AVS.

Plasma metanephrine for assessing the selectivity of adrenal venous sampling

Introduction: Adrenal vein sampling (AVS) is used to distinguish unilateral from bilateral causes of primary aldosteronism. More reliable parameters than cortisol are needed for assessing correct catheter positioning during AVS. The object of this study was to determine the utility of adrenal venous (AV) plasma concentrations of metanephrine to establish correct catheter positioning during AVS.

Participation of adrenal medulla in 6-amino-nicotinamide-induced hyperglycaemia in the rat.

In fasted rats 6-aminonicotinamide (6-AN) produced delayed hyperglycaemia, the peak effect being seen by 7 h. Fasting plasma insulin concentrations were not significantly altered but liver glycogen concentrations were decreased following treatment with 6-AN. Adrenalectomy, demedullation and pretreatment with reserpine, phentolamine, nicotinamide and nicotinic acid completely blocked the hyperglycaemic response whereas guanethidine and propranolol or oxprenolol were ineffective. Catecholamine concentrations in the adrenal venous plasma were markedly increased by treatment with 6-AN, the peak effect being seen by 5 h. It is concluded that adrenal medullary release, which is slow in onset, is mainly responsible for the development of sustained hyperglycaemia in the rat.

Identification and quantification of diadenosine polyphosphate concentrations in human plasma.

Diadenosine polyphosphates have been demonstrated to be involved in the control of vascular tone as well as the growth of vascular smooth muscle cells and hence, possibly, in atherogenesis. In this study we investigated the question of whether diadenosine polyphosphates are present in human plasma and whether a potential source can be identified that may release diadenosine polyphosphates into the circulation. Plasma diadenosine polyphosphates (ApnA with n=3 to 6) were purified to homogeneity by affinity-, anion exchange-, and reversed phase-chromatography from deproteinized human plasma. Analysis of the homogeneous fractions with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed molecular masses ([M+H]+) of 757, 837, 917, and 997 d. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrometry mass spectra of these fractions with those of authentic diadenosine polyphosphates revealed that these isolated substances were identical to Ap3A, Ap4A, Ap5A, and Ap6A. Enzymatic analysis showed an interconnection of the phosphate groups with the adenosines in the 5'-positions of the ribose moieties. The mean total plasma diadenosine polyphosphate concentrations (micromol/L; mean +/- SEM) in cubital veins of normotensive subjects amounted to 0.89+/-0.59 for Ap3A, 0.72+/-0.72 for Ap4A, 0.33+/-0.24 for Ap5A, and 0.18+/-0.18 for Ap6A. Cubital venous plasma diadenosine polyphosphate concentrations from normotensives did not differ significantly from those in the hypertensive patients studied. There was no significant difference between arterial and venous diadenosine polyphosphate plasma concentrations in 5 hemodialysis patients, making a significant degradation by capillary endothelial cells unlikely. Free plasma diadenosine polyphosphate concentrations are considerably lower than total plasma concentrations because approximately 95% of the plasma diadenosine polyphosphates were bound to proteins. There were no significant differences in the diadenosine polyphosphate plasma concentrations depending on the method of blood sampling and anticoagulation, suggesting that platelet aggregation does not artificially contribute to plasma diadenosine polyphosphate levels in significant amounts. The ApnA (with n=3 to 6) total plasma concentrations in adrenal veins were significantly higher than the plasma concentrations in both infrarenal and suprarenal vena cava: adrenal veins: Ap3A, 4.05+/-1.63; Ap4A, 6.18+/-2.08; Ap5A, 0.53+/-0.28; Ap6A, 0.59+/-0.31; infrarenal vena cava: Ap3A, 1.25+/-0.66; Ap4A, 0.91+/-0.54; Ap5A, 0.25+/-0.12; Ap6A, 0.11+/-0.06; suprarenal vena cava: Ap3A, 1.40+/-0.91; Ap4A, 1.84+/-1.20; Ap5A, 0.33+/-0.13; Ap6A, 0.11+/-0.07 (micromol/L; mean +/- SEM; each P<0.05 (concentration of adrenal veins versus infrarenal or suprarenal veins, respectively). The presence of diadenosine polyphosphates in physiologically relevant concentrations in human plasma was demonstrated. Because in adrenal venous plasma significantly higher diadenosine polyphosphate concentrations were measured than in plasma from the infrarenal and suprarenal vena cava, it can be assumed that, beside platelets, the adrenal medulla may be a source of plasma diadenosine polyphosphates in humans.

Effect of angiotensin II on aldosterone secretion in canine adrenal gland in situ.

To investigate the effect of angiotensin (ANG) II on aldosterone (ALDO) secretion, we measured arterial and adrenal venous plasma aldosterone concentrations in anesthetized dogs. The intraadrenal arterial infusion of ANG II (0.3 ng/kg/min) or potassium chloride (KCl) (0.6 mg/min) increased ALDO secretion. The changes in ALDO secretion in response to ANG II were tested during the concomitant arterial infusion of two graded doses of losartan (10 and 100 ng/kg/min), PD 123319 (50 and 500 ng/kg/min), nifedipine (25 and 250 ng/kg/min), or TMB-8 (2 and 20 microg/kg/min). All of these test drugs except PD123319 inhibited the ANG II-induced increase in ALDO secretion. Losartan did not affect the KCl-induced increase in ALDO secretion. These results indicate that ANG II acts on ANG II type 1 receptors in the adrenal gland and enhances ALDO secretion. They also suggest the involvement of both intracellular and extracellular calcium in the aldosterone response to stimulation by ANG II. Under these in vivo experimental conditions, the KCl-stimulated ALDO secretion does not appear to involve ANG II formation in the adrenal gland.

The adrenal glands as suppliers of plasma L-Dopa and sources of urinary dopamine.

Dopamine (DA) is a natriuretic hormone synthesized in the kidneys by conversion of filtered 3,4-dihydroxyphenylalanine (L-Dopa), and is activated during hypervolaemia and increased dietary sodium intake. The natriuretic activity of endogenous DA is controversial, however, and the regulation of renal DA synthesis has yet to be explained. It has been suggest that the adrenals may be major suppliers of plasma L-Dopa on the basis of their catecholamine biosynthesis. A study was conducted in rats to elucidate the role of the adrenal glands as dynamic suppliers of L-Dopa to plasma, and thereby as sources of urinary DA. Adrenal venous and systemic arterial plasma concentrations and urinary excretion of L-Dopa, DA and sodium were measured before and during acute isotonic volume expansion (VE; 5% of body weight). One group of animals were acutely adrenalectomized (ADX group) to elucidate the ultimate importance of the adrenals in VE-induced renal sodium and DA excretion. In intact animals, the L-Dopa concentration was 62% higher in adrenal venous than in systemic arterial plasma under control conditions, and 42% higher during VE. The adrenaline concentration was 65 times higher in adrenal venous than in systemic arterial plasma before VE and 56 times higher during VE. The L-Dopa concentration in systemic arterial plasma and the urinary L-Dopa excretion were similar in intact and ADX animals. In intact animals, renal sodium and DA excretion during VE increased more than 13-fold and by 42%, respectively. The corresponding values in ADX animals did not differ from those in the intact animals (more than 14-fold and 36%, respectively). It is concluded that the adrenal glands are only minor suppliers of plasma L-Dopa and minor sources of urinary DA. The regulation of plasma L-Dopa remains to be explained.

Effects of Intracerebroventricular Injected Choline on Cardiovascular Functions and Sympathoadrenal Activity

Intracerebroventricular (i.c.v.) injection of choline (50-150 micrograms) increased blood pressure (SP) and decreased heart rate (HR) in freely moving rats. Intracerebroventricular pretreatment of rats with mecamylamine (50 micrograms) blocked the reduction in HR and reduced the increase in SP induced by i.c.v. choline (150 micrograms). Central muscarinic blockade with atropine (10 micrograms, i.c.v.) reduced the pressor response to i.c.v. choline (150 micrograms) by about 70%, without influencing the decrease in HR. The decrease in HR induced by i.c.v. choline was prevented by intraarterial (i.a.) treatment of atropine methylnitrate (2 mg/kg). Intracerebroventricular choline (150 micrograms) produced a fivefold increase in catecholamine concentrations in adrenal venous plasma. Bilateral adrenalectomy reduced, but did not block, choline's effect on SP. Intracerebroventricular choline (150 micrograms) showed an ability to increase and restore SP in rats subjected to spinal cord transection or pretreatment with hexamethonium (15 mg/kg, i.a.) or with phentolamine (10 mg/kg, i.a.). Intracerebroventricular choline (150 micrograms) increased plasma vasopressin (VP) levels from 2.2 +/- 0.4 to 25.6 +/- 2.5 pg/ml. Pretreatment of rats with a VP antagonist reduced the pressor response to i.c.v. choline. It is concluded that (a) the reduction in HR results from a central nicotinic receptor-mediated increase in vagal tone, (b) the increase in SP appears to be due to activation of both nicotinic and muscarinic central cholinergic receptors, and that (c) the central activation of the adrenal medulla and the increase in plasma levels of VP are involved in the pressor response to i.c.v. choline.

Identification of 5β-pregnane and 5β-androstane derivatives in adrenal venous and peripheral blood plasma of the female possum ( Trichosurus vulpecula)

In the possum a marked sex difference has been found in the steroids in adrenal venous plasma. Four 5β-pregnane and four 5α(β) androstane derivatives together with ten 4-ene-3-keto steroids were isolated from the adrenal venous plasma of the female and definitively identified by gas chromatography-mass spectrometry. The major reduced steroids were: 5β-pregnane-3α,17α-diol-20-one and 5β-pregnane-3α,17α,20α-triol, at concentrations of 52 ± 12 μg/100 ml and 44 ± 8 μg/100 ml mean ± SEM respectively. The concentration of cortisol was 198 ± 47 μg/100 ml. The concentration of the 2 reduced steroids in peripheral plasma were approx. 100 times less. In contrast the adrenal venous plasma of a male contained 14 steroids of which only three, found in trace amounts, were reduced. The results confirm previous in vitro observations that reduced steroids are produced by the adrenocortical special zone, which is only present in the female. The physiological significance of the presence of reduced steroids of adrenocortical origin in the circulation of the female possum is discussed.

Identification of 5α-androstane-3α,17α-diol in adrenal venous plasma of female possum ( Trichosurus vulpecula)

Two hitherto unidentified C19 steroids were isolated from adrenal venous blood plasma of female possum ( Trichosurus vulpecula). They were 5α-androstane-3α,17α-diol and its isomer 5β-androstane-3α,17α-diol. The compounds were isolated and identified by fractionation on paper chromatograms and HPLC, followed by gas chromatography-mass spectrometry after conversion to trimethylsilyl ethers. In adrenal venous plasma the concentrations of the 5α-isomer ranged from 24–71 μg/100 ml, 47.5 ± 7.7 (mean ± SEM)s and of the 5β-isomer 1.5–9.3 μg/100 ml, 5.6 ± 1.6 (mean ± SEM). In peripheral plasma only the 5α-derivative was detectable, the highest concentration being 0.97 μg/100 ml. The reduced steroids were not detected in the plasma of a male possum, confirming previous in vitro evidence that reduced steroids are products of the adrenocortical special zone which is found only in the female.

Secretion of corticotrophin releasing factor from the adrenal during splanchnic nerve stimulation in conscious calves.

1. The output of corticotrophin releasing factor-like immunoreactivity (CRF) from the adrenal gland has been investigated using the 'adrenal clamp' technique in conscious calves. 2. Stimulation of the peripheral end of the splanchnic nerve for 10 min increased the mean output of CRF progressively, so that it had risen by about twentyfold, to a peak incremental value of 24 +/- 4 pmol min-1 kg-1 at 10 min. This response was significantly increased by stimulating in bursts at 40 Hz for 1 s at 10 s intervals, which raised the mean CRF output by 44 +/- 7 pmol min-1 kg-1 at 10 min (P less than 0.05). 3. The mean output of adrenaline and noradrenaline rose more abruptly in response to splanchnic nerve stimulation with peak incremental values realized within 2.5 min. However, the ratios of adrenal CRF to catecholamine output were closely similar during the later stages of stimulation (7.5-10 min). There was a similarly abrupt rise in adrenal cortisol output in response to splanchnic nerve stimulation which was, nevertheless, linearly related to arterial plasma ACTH concentration throughout. 4. In hypophysectomized calves, administration of adrenocorticotrophic hormone (ACTH1-24) at a dose of 5 ng min-1 kg-1 reduced the output of adrenal CRF in response to splanchnic nerve stimulation by about 50% (P less than 0.05). 5. CRF isolated from adrenal venous effluent plasma, collected both at rest and during splanchnic nerve stimulation, was separated by reverse-phase high-pressure liquid chromatography and found to elute in a position identical to that of human 41CRF. This suggests that adrenal CRF is structurally closely similar to its pituitary counterpart.

The adrenal contribution to the neuroendocrine responses to splanchnic nerve stimulation in conscious calves.

1. The extent to which the adrenal gland contributes to neuroendocrine responses to electrical stimulation of the peripheral end of the splanchnic nerve has been investigated in conscious calves in which the right nerve was stimulated either at 4 Hz continuously for 10 min or at 40 Hz in 1 s bursts at 10 s intervals for the same period. 2. It was confirmed that the release of neuropeptide Y (NPY) and of gastrin-releasing peptide (GRP) is potentiated by stimulation in bursts at a relatively high frequency and shown that the adrenal gland made a negligible contribution to these responses. 3. There was no detectable change in the concentration of vasoactive intestinal peptide (VIP) in the arterial plasma but the existence of a very small but highly significant rise in the output of VIP from the adrenal provided evidence that it was released within the gland in response to splanchnic nerve stimulation. 4. The concentration of calcitonin gene-related peptide (CGRP) in the arterial and adrenal venous effluent plasma was consistently below the level of detection of the assay. 5. Splanchnic nerve stimulation resulted in an abrupt rise in the output of both free and total met5-enkephalin-like immunoreactivity from the adrenal gland which was substantially potentiated by stimulating in bursts. This pattern of stimulation also increased the proportion released in a high-molecular-weight form. 6. Stimulation in bursts significantly enhanced the output of both adrenaline and noradrenaline from the adrenal and resulted in the release of proportionately more noradrenaline. Small amounts of dopamine and DOPAC were also released during splanchnic nerve stimulation and the output of dopamine was significantly increased by stimulating in bursts. 7. Both patterns of stimulation elicited an abrupt rise in mean plasma adrenocorticotrophic hormone (ACTH) concentration, which was associated with an increase in mean adrenal cortisol output and the former effect was significantly enhanced by stimulating in bursts. 8. It is concluded that certain responses to splanchnic nerve stimulation are significantly potentiated by an intermittent high-frequency pattern of stimulation, including all those that are attributable to adrenal medullary activity, whereas others are apparently unaffected by changes in stimulus pattern.

Adrenal secretion of epinephrine after stimulation of trigeminal nucleus caudalis depends on stimulus pattern.

Adrenal catecholamine secretion after stimulation of trigeminal nucleus caudalis (Vc) was compared to that evoked from more ventrolateral brain stem areas (VLM) by sampling adrenal venous blood in anesthetized cats. The effect of stimulus pattern on catecholamine secretion was assessed by presenting an equal number (300 pulses over 15 s) of electrical stimuli (75 microA, 0.2 ms) as a continuous pattern and as a burst pattern pulse train at each electrode site. Epinephrine secretion increased promptly by 1 min after burst pattern stimulation of Vc, whereas continuous pattern stimuli had no consistent effect. Burst pattern stimulation of VLM sites caused a small decrease in epinephrine secretion, whereas continuous pattern stimuli had no significant effect. Adrenal norepinephrine increased equally after Vc or VLM stimulation regardless of stimulus pattern. The adrenal catecholamine secretory responses could not be explained by the transient evoked changes in adrenal venous plasma flow or arterial pressure. The data indicate that stimulation of Vc evokes a pattern-dependent increase in epinephrine secretion, whereas the increase in norepinephrine secretion is equal after burst or continuous pattern stimuli. Further, adrenal activation by brief neural stimuli cannot be assessed adequately in the cat by measuring epinephrine secretion alone, since stimulation of many ventrolateral brain stem sites caused a significant increase in norepinephrine secretion with no corresponding increase in epinephrine secretion.

Corticotropin-Releasing Factor in the Dog Adrenal Medulla Is Secreted in Response to Hemorrhage*

To characterize the nature of CRF-like immunoreactivity (CRF-LI) in the dog adrenal, adrenal medullary, adrenal cortical, or hypothalamic tissue was extracted and subjected to RIA after partial purification on C-18 cartridges or Sephadex G-50. Using N- and C-terminal-directed antisera against rat/human (r/h) CRF, significant levels of CRF-LI were found in the adrenal medulla and hypothalamus, but not in the adrenal cortex. Immunocytochemical analysis revealed that CRF-immunoreactive cells were located in the adrenal medulla, many of them concentrated in the vicinity of blood vessels and at the border between adrenal medulla and cortex. However, the cortex was devoid of any CRF-positive cells. On reverse phase HPLC, CRF-LI in the adrenal medulla coeluted with synthetic r/hCRF. In a bioassay system, using dispersed rat anterior pituitary cells, purified adrenal CRF caused a dose-dependent increase in ACTH secretion parallel to the r/hCRF standard, indicating that dog adrenal medulla contains authentic r/hCRF. Evidence of CRF-LI secretion from the adrenal was supported by its presence in adrenal venous, but not in peripheral arterial, plasma. Adrenal venous plasma CRF-LI coeluted with r/hCRF on reverse phase HPLC after affinity chromatographic purification. The CRF-LI secretory rate in conscious trained dogs was 68 +/- 19 pg/min (concentration, 27 +/- 5 pg/ml). In response to 20% hemorrhage, the CRF-LI secretion rate rose 3-fold within 15 min and was associated with increased catecholamine secretion. The existence of a biologically active CRF-like substance in the dog adrenal medulla and its secretion in conjunction with catecholamines after a hemorrhage suggest a physiological role for this peptide other than pituitary or central nervous system regulation.

Biochemical characterization of circulating Met-enkephalins in canine endotoxin shock.

The alterations in arterial, venous and adrenal vein levels of immunoreactive Met-enkephalins following endotoxin administration have been investigated in dogs by direct measurement and gel filtration chromatography. Animals were anaesthetized with alpha-chloralose and allowed to breath spontaneously. The left lumbar adrenal vein, limb vein and femoral artery were cannulated for blood sampling. Severe shock was produced by the administration of a large bolus of E. coli endotoxin followed by a continuous infusion. The production of endotoxin shock was associated with significant increases in adrenal vein and systemic venous plasma immunoreactive Met-enkephalin levels. Forty-five minutes after induction of endotoxin shock arterial immunoreactive Met-enkephalin levels were generally higher than baseline values. In resting anaesthetized animals a large 31,000 molecular weight form of Met-enkephalin, presumably proenkephalin, was found in plasma obtained from the adrenal vein, systemic and pulmonary circulations. Following endotoxin this enkephalin-containing peptide still predominated in arterial and venous plasma, whereas in the adrenal vein the proportion of Met-enkephalin immunoreactivity attributable to this large peptide fell. This was associated with the appearance of increasing amounts of smaller molecular forms (18,000, 8000, 3-5000 molecular weight and the pentapeptide itself). In this model enkephalin-containing peptides were not biochemically modified by their passage through the lungs.

Co-release of neuropeptide Y and catecholamines upon adrenal activation in the cat.

The release of neuropeptide Y (NPY)-like immunoreactivity (-LI) in relation to catecholamines from the cat adrenal was studied in anaesthetized animals. Abdominal surgery increased plasma levels of NPY-LI from 65 +/- 6 to 149 +/- 26 pmol l-1. A positive veno-arterial concentration gradient over the adrenal gland was found for both NPY-LI, adrenaline (Adr) and noradrenaline (NA) during basal conditions. Asphyxia for 2 min increased the output of both NPY-LI and catecholamines from the adrenal. Electrical stimulation of the splanchnic nerve caused a marked increase in adrenal output of NPY-LI and catecholamines. The adrenal content of NPY-LI, as well as the release of NPY-LI from the adrenal, was at least 1000-fold lower on a molar basis than that of catecholamines. The concentration of NPY-LI in the adrenal vein upon splanchnic nerve stimulation was in the nM range. Reversed-phase HPLC characterization revealed that NPY-LI in the adrenal, and in the adrenal venous plasma collected during splanchnic nerve stimulation, was closely related to synthetic porcine NPY. Stimulation with bursts of 20 Hz for 1 S with 10 s intervals for 2 min caused a four-fold higher output of NPY-LI and Adr compared to a continuous stimulation with 2 Hz, giving the same number of impulses. The NA output, however, was only slightly increased by burst stimulation. Guanethidine did not reduce the adrenal output of NPY-LI or catecholamines induced by splanchnic nerve stimulation, while the release was abolished by chlorisondamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma catecholamines associated with hypothalamically-elicited defense behavior

Sympatho-adrenal (SA) activation was determined by measuring levels of norepinephrine (NE) and epinephrine (E) in bilateral adrenal venous and peripheral venous plasma of 20 anesthetized cats following stimulation of medial hypothalamic sites. Hypothalamic sites were selected that elicited affective defense behavior in the freely moving cat. Fifty-eight percent of these hypothalamic sites elicited a bilateral increase ⩾ 10 ng/min in the output of both adrenal catecholamines (CAs); these increases were greater from the gland ipsilateral to the side of stimulation. Other SA responses included both preferential increases or decreases in either NE or E. Under baseline conditions, an average of 67% of the NE in the peripheral venous plasma was contributed by the sympathetic noradrenergic nerves; hypothalamic stimulation at “defense” sites increased the contribution to 75%. The data suggest that hypothalamic regions that elicit defense behavior may overlap with regions that activate the adrenal medullary and sympathetic nervous system.