Lucie Chouinard

VASOPRESSIN : A POTENT AUTOCRINE/PARACRINE REGULATOR OF MAMMAL ADRENAL FUNCTIONS.

The control of adrenal functions by locally secreted neuropeptides or neurotransmitters is of great physiological importance. Vasopressin (VP) is one of these autocrine/paracrine regulators. We demonstrated by RT-PCR and perifusion experiments that rat and human adrenal medulla expressed and released vasopressin under basal conditions and under stimulation by acetylcholine. Intra-adrenal concentrations of VP may be sufficient to activate adrenal VP receptors. In the cortex, only the V1a receptor subtype has been detected. It triggered both steroid secretion and cortical growth. In the medulla, both V1a and V1b receptor subtypes were expressed. V1b receptors were mainly present on chromaffin cells and stimulated catecholamine secretion. The role of the V1a receptor remains unclear. Pathophysiological studies also revealed that human pheochromocytoma did not overexpress vasopressin receptors but might oversecrete vasopressin causing high plasma VP concentrations and elevated blood pressure.

Proven infection-related sepsis induces a differential stress response early after ICU admission

IntroductionNeuropeptides arginine-vasopressin (AVP), apelin (APL), and stromal-derived factor-1α (SDF-1α) are involved in the dysfunction of the corticotropic axis observed in septic ICU patients. Study aims were: (i) to portray a distinctive stress-related neuro-corticotropic systemic profile of early sepsis, (ii) to propose a combination data score, for aiding ICU physicians in diagnosing sepsis on admission.MethodsThis prospective one-center observational study was carried out in a medical intensive care unit (MICU), tertiary teaching hospital. Seventy-four out of 112 critically ill patients exhibiting systemic inflammatory response syndrome (SIRS) were divided into two groups: proven sepsis and non sepsis, based on post hoc analysis of microbiological criteria and final diagnosis, and compared to healthy volunteers (n = 14). A single blood sampling was performed on admission for measurements of AVP, copeptin, APL, SDF-1α, adrenocorticotropic hormone (ACTH), cortisol baseline and post-stimulation, and procalcitonin (PCT).ResultsBlood baseline ACTH/cortisol ratio was lower and copeptin higher in septic vs. nonseptic patients. SDF-1α was further increased in septic patients vs. normal patients. Cortisol baseline, ACTH, PCT, APACHE II and sepsis scores, and shock on admission, were independent predictors of sepsis diagnosis upon admission. Using the three first aforementioned categorical bio-parameters, a probability score for predicting sepsis yielded an area under the Receiver Operating Curve (ROC) curves better than sepsis score or PCT alone (0.903 vs 0.727 and 0.726: P = 0.005 and P < 0.04, respectively).ConclusionsThe stress response of early admitted ICU patients is different in septic vs. non-septic conditions. A proposed combination of variable score analyses will tentatively help in refining bedside diagnostic tools to efficiently diagnose sepsis after further validation.

Music and biological stress dampening in mechanically-ventilated patients at the intensive care unit ward—a prospective interventional randomized crossover trial ☆ ☆☆

PURPOSEnTo evaluate the impact of slow-tempo music listening periods in mechanically ventilated intensive care unit patients.nnnMETHODSnA randomized crossover study was performed in a 16-bed, adult critical care unit at a tertiary care hospital. Still-sedated patients, mandating at least 3 more days of mechanical ventilation, were included. The intervention consisted in two 1-hour daily periods of music-vs-sham-MP3 listening which were performed on Day 1 or 3 post-inclusion, with a Day 2 wash-out. Before-after collection of vital signs, recording of daily sedative drug consumption and measurement of stress and inflammatory blood markers were performed.nnnRESULTSnOf 55 randomized patients, 49 were included in the final analyses. Along with music listening, (i) vital signs did not consistently change, whereas narcotic consumption tended to decrease to a similar sedation (P = .06 vs sham-MP3); (ii) cortisol and prolactin blood concentrations decreased, whereas Adreno Cortico Trophic Hormone (ACTH)/cortisol ratio increased (P = .02; P = .038; and P = .015 vs sham-MP3, respectively), (iii) cortisol responders exhibited reversed associated changes in blood mehionine (MET)-enkephalin content (P = .01).nnnCONCLUSIONSnIn the present trial, music listening is a more sensitive stress-reliever in terms of biological vs clinical response. The hypothalamus-pituitary adrenal axis stress axis is a quick sensor of music listening in responding mechanically ventilated intensive care unit patients, through a rapid reduction in blood cortisol.

Involvement of tyrosine phosphorylation and mapk activation in the mechanism of action of acth, angiotensin II and vasopressin

ACTH, Angiotensin II (Ang II) and Vasopressin (AVP) are among the well known regulators of aldosterone secretion and also have a trophic action on the adrenal gland. According to classic studies, Ang II and AVP activate phospholipase C (PLC), diacylglycerol (DAG) and inositol phosphate (InsPs) production whereas ACTH activates cAMP production. However, our data indicate that the three peptides are able to induce a time-dependent increase in the level of Tyr-phosphorylation of several proteins. Western Blot analysis indicates a biphasic activation of Tyr-phosphorylation by AVP, with a peak at 30 s and a second one at 15 min incubation. Ang II induced a rapid (2 min) and sustained activation of Tyr-phosphorylation, while ACTH induced a progressive time course with a plateau reached at 15 min. Ang II and AVP also increased phosphorylation of p42mapk and p44mapk, while ACTH did not affect MAPK activity. Moreover, pre-incubation of the cells with genistein (Tyr-kinase inhibitor) and PD 098059 (a MAPK inhibitor) did not affect InsPs production or aldosterone secretion induced by Ang II or AVP. These results suggest that the MAPK pathway is involved in the control of cell growth rather than aldosterone secretion.

Bioinactive ACTH Causing Glucocorticoid Deficiency

CONTEXTnA 4-year-old girl and a 4-month-old boy presented with hypoglycemia, normal electrolytes, low cortisol, and high ACTH. A diagnosis of primary adrenal insufficiency was made and initial treatment was with glucocorticoids and mineralocorticoids. The genes known to cause ACTH resistance were normal. Whole exome sequencing revealed that the girl was compound heterozygous for POMC mutations: one previously described null allele and one novel p.R8C mutation in the sequence encoding ACTH and α-MSH. The boy was homozygous for the p.R8C mutation.nnnHYPOTHESISnThe p.R8C ACTH mutant is immunoreactive, but the mutant peptides, ACTH-R8C and α-MSH-R8C, are bioinactive.nnnMETHODSnMethods included whole exome sequencing, Sanger sequencing, peptide synthesis, ACTH immunoradiometric assay, hormone binding, and activation assays in cells expressing melanocortin receptors.nnnRESULTSnACTH-R8C was immunoreactive but failed to bind and activate cAMP production in melanocortin-2 receptor (MC2R)-expressing cells, and α-MSH-R8C failed to bind and stimulate cAMP production in MC1R- and MC4R-expressing cells.nnnCONCLUSIONnThese are the first documented cases of glucocorticoid deficiency due to the secretion of an ACTH molecule that lacks biological bioactivity but conserves immunoreactivity. POMC mutations should thus be considered in patients presenting with apparent ACTH resistance. Our findings also highlight a limitation to immunoassay-based diagnostics and demonstrate the value of genetic analysis. Establishing the molecular etiology of the disorder in our patients allowed cessation of the unnecessary mineralocorticoids. Finally, discovery of this mutation indicates that in humans, the amino acid sequence His(6)Phe(7)Arg(8)Trp(9) is important not only for cAMP activation but also for ACTH binding to MC2R.

A model for studying regulation of aldosterone secretion: Freshly isolated cells or cultured cells?

Practically all studies relating to zona glomerulosa function have been performed either with freshly isolated cells or with cells used after 2 or 3 days in culture. This study compares the step-by-step response (binding, second messenger production and aldosterone response) of isolated glomerulosa cells vs cells maintained in primary culture to the main stimuli of aldosterone secretion. One day in culture induces a decrease of 77 and 65% in the basal level of corticosterone and aldosterone secretions, compared to that observed in freshly isolated cells. In these conditions, the cells become more sensitive to most of their stimuli, but not all: e.g. important differences are noted in the dose-response of aldosterone secretion to adrenocorticotropin (ACTH), which is often shifted to a lower concentration sensitivity in cultured cells. For example, 0.1 nM ACTH stimulates steroid secretion by three-fold in isolated cells while 1 pM ACTH already induces a 25 and nine-fold increase, respectively, in corticosterone and aldosterone output in cultured cells. Moreover, some stimuli such as isoproterenol do not have any effect in isolated cells but do stimulate steroid secretion in cultured cells. In contrast, other stimuli, such as serotonin or DA (via DA2 receptors) act preferentially in freshly isolated cells. The main observation derived from this study is that glomerulosa cells, under appropriate conditions, are able to respond to their main secretagogues even after 4 days in culture. At this time, glomerulosa cells maintain their ultrastructural characteristics and functional properties and, aside from a few exceptions, demonstrate higher sensitivity to their known stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms involved in the interaction of dopamine with angiotensin II on aldosterone secretion in isolated and cultured rat adrenal glomerulosa cells.

In a previous study, we have shown that freshly isolated glomerulosa cells possess dopamine (DA) receptors from both DA-1 and DA-2 subclasses, whereas in cultured conditions, cells exhibit dopamine receptors from the DA-1 subclass only. In the present work, we have studied the effect of DA on angiotensin-stimulated glomerulosa cells in these two experimental conditions. Our results demonstrate that in isolated cells, angiotensin II (AT) stimulates inositol phosphate accumulation, calcium influx and steroid secretion. Treatment with pertussis toxin completely blocks AT-stimulated steroid secretion and calcium influx and partially reduces inositol phosphate accumulation. DA alone has no effect on cAMP accumulation. However, in the presence of a specific DA-1 antagonist (SCH 23390), DA reduces intracellular cAMP content. Similarly, DA-like pertussis toxin produces the same inhibitory effects on AT-stimulated cells. The combined influence of DA and pertussis toxin is not additive suggesting that a Gi GTP-binding protein is involved in the DA action. Specific DA antagonists indicate that these inhibitory processes are mediated through the DA-2 receptor subtype. DA may act by decreasing the intracellular calcium concentration since it reduces AT-stimulated Ca2+ influx and that both phospholipase C (PLC) and steroid accumulation are calcium dependent. Yet a direct inhibitory coupling between the DA-2 receptor and PLC may represent a second alternative since DA inhibitory effects are always present when calcium influx is artificially increased or decreased. In cultured cells, we observe an additive effect of DA and AT on aldosterone secretion, which is the result of additive interactions of the second messengers involved, namely cAMP for dopamine and inositol phosphates for angiotensin II. From these studies, we conclude that DA may exert a more versatile effect on aldosterone secretion than previously suspected.

Adrenocorticotropin hormone (ACTH) effects on MAPK phosphorylation in human fasciculata cells and in embryonic kidney 293 cells expressing human melanocortin 2 receptor (MC2R) and MC2R accessory protein (MRAP)β

Adrenocorticotropin hormone (ACTH) exerts trophic effects on adrenocortical cells. We studied the phosphorylation of mitogen-activated proteins kinases (MAPKs) in human embryonic kidney cells stably expressing the ACTH receptor, MC2R, and its accessory protein MRAPβ and in primary cultures of human adrenal fasciculata cells. ACTH induced a maximal increase in p44/p42(mapk) and of p38 MAPK phosphorylation after 5min. Neither the overexpression of wild-type arrestin2, arrestin3 or their respective dominant negative forms affected p44/p42(mapk) phosphorylation. However, preincubation with the recycling inhibitors brefeldin A and monensin attenuated both cAMP accumulation and p44/p42(mapk) phosphorylation proportionally. Cyclic AMP-related PKA inhibitors (H89, KI(6-22)) and Rp-cAMPS decreased p44/p42(mapk) phosphorylation but not ACTH-mediated cAMP production. The selective Epac1/2 activator, 8-pCPT-2-O-MecAMP, did not modify the effect of ACTH. Thus, cAMP/PKA, but not cAMP/Epac1/2 pathways, or arrestin-coupled internalization of MC2R is involved in ACTH-induced p44/p42(mapk) phosphorylation by human MC2R. Together, ACTH binding to MC2R stimulates PKA-dependent p44/p42(mapk) phosphorylation.

Dual effects of dopamine in rat adrenal glomerulosa cells

The effects of dopamine (DA) on cAMP production and aldosterone secretion were compared in freshly isolated cells and in primary cultures of rat adrenal glomerulosa cells. Under isolated conditions, glomerulosa cells exhibited dopamine receptors from DA-1 and DA-2 subclass, whereas in cultured conditions, where cells are very sensitive to their known stimuli, cells only exhibited dopamine receptors from the DA-1 subclass. Moreover, unlike freshly isolated cells, dopamine stimulated both cAMP production and aldosterone secretion in 3-day cultured preparations. These effects were receptor specific since they were completely suppressed by Scherring 23390 (a specific DA-1 antagonist) and were unaffected by a beta-adrenergic antagonist. As in vivo rat adrenal cortex contains DA, we discuss a possible involvement of this neurotransmitter in the regulation of aldosterone secretion.

Postprandial fatty acid uptake and adipocyte remodeling in angiotensin type 2 receptor-deficient mice fed a high-fat/high-fructose diet

ABSTRACT The role of the angiotensin type-2 receptor in adipose physiology remains controversial. The aim of the present study was to demonstrate whether genetic angiotensin type-2 receptor-deficiency prevents or worsens metabolic and adipose tissue morphometric changes observed following a 6-week high-fat/high-fructose diet with injection of a small dose of streptozotocin. We compared tissue uptake of nonesterified fatty acid and dietary fatty acid in wild-type and angiotensin type-2 receptor-deficient mice by using the radiotracer 14(R,S)-[18F]-fluoro-6-thia-heptadecanoic acid in mice fed a standard or high-fat diet. Postprandial fatty acid uptake in the heart, liver, skeletal muscle, kidney and adipose tissue was increased in wild-type mice after a high-fat diet and in angiotensin type-2 receptor-deficient mice on both standard and high-fat diets. Compared to the wild-type mice, angiotensin type-2 receptor-deficient mice had a lower body weight, an increase in fasting blood glucose and a decrease in plasma insulin and leptin levels. Mice fed a high-fat diet exhibited increased adipocyte size that was prevented by angiotensin type-2 receptor-deficiency. Angiotensin type-2 receptor-deficiency abolished the early hypertrophic adipocyte remodeling induced by a high-fat diet. The small size of adipocytes in the angiotensin type-2 receptor-deficient mice reflects their inability to store lipids and explains the increase in fatty acid uptake in non-adipose tissues. In conclusion, a genetic deletion of the angiotensin type-2 receptor is associated with metabolic dysfunction of white adipose depots, and indicates that adipocyte remodeling occurs before the onset of insulin resistance in the high-fat fed mouse model.