Norepinephrine Responses Research Articles

Protein-Protein Interactions of the β-adrenergic Receptor and Kinetic Analysis of Intracellular Binding Partners via µ-Patterned Surfaces

β-adrenergic (βAR) receptors belong to the class of G protein-coupled receptors (GPCRs) and generally administrate the physiological response of adrenaline and noradrenaline. They can be classified into three receptor subtypes (β1, β2, β3) which are major drug targets for some of the most commonly prescribed drugs in the history of medicine. The various βAR subtypes also interact differentially with a huge diversity of cytoplasmic (G-proteins, arrestin, adenylate cyclase,⋯) and transmembrane proteins (e.g. receptor homo-/hetero-multimerization, ion channels,⋯) leading to distinctive receptor signaling pathways. Studying such interaction processes of plasma-membrane localized receptor proteins is key for a better understanding of cellular processes. In the past few years, many studies indicated that βAR dimerization can occur between two identical receptors (homodimerization), between two different receptor subtypes of the same family or between receptors of different families (heterodimerization). The physiological role of such di-/multimerizations remains a matter of debate. Here we describe an assay combining TIR (total internal reflection) microscopy and micro-patterned surfaces, which can be used for the detection of protein-protein interactions in and near the cell membrane in-vivo. We studied the homo- and heteromultimerization of β1- and β2-adrenergic receptors. Furthermore we investigated the interaction of βARs with different cytoplasmic signaling proteins such as β-arrestin, G-protein and adenylate cyclase. We unequivocally show that β1ARs as well as β2ARs form homo- and hetero multimers in living CHO-K1 cells. We could also proof the interaction of βARs with β-arrestin, G-protein and adenylate cyclase. In addition we conducted FRAP (fluorescence after photobleaching) experiments for further characterization of these interaction processes. In general, our system is of great interest for a fast and straightforward analysis of membrane-receptor protein interactions.

Running training attenuates blood pressure and norepinephrine responses to immobilization stress in spontaneously hypertensive rats

Running training attenuates blood pressure and norepinephrine responses to immobilization stress in spontaneously hypertensive rats

Vascular reactivity of arteria femoralis in adult and aged spontaneously hypertensive and Wistar-Kyoto rats

The relationship of age and hypertension on endothelial dysfunction and increased responses to vasoconstrictor stimuli. Hypertension is a disease accompanied by endothelial dysfunction and is characterized by an impaired vascular reactivity and enhanced activity of sympathetic nervous system. In our experiment, we used spontaneously hypertensive rats representing model of essential hypertension and the Wistar-Kyoto rats as normotensive strain. Femoral arteries of adult and aged rats were put into the chamber of Mulvany-Halpern isometric myograph. As the nutrient solution, the modified Krebs-Henseleit solution having temperature 37 °C and bubbled with O2 was used. After 30 minutes stabilization of blood vessels, a dose-dependent curve of norepinephrine response was recorded (concentrations 3x10-8 M, 10-7 M, 3x10-7 M, 10-6 M, 3x10-6 M, 10-5 M, 3x10-5 M, 10-4 M), followed by a dose-dependent curve of acetylcholine response (concentrations 3x10-8 M, 10-7 M, 3x10-7 M, 10-6 M, 3x10-6 M). Our experiments recorded an increased reactivity to contraction stimuli in spontaneously hypertensive animals. Vascular reactivity to norepinephrine at 5 month and 12 month old rats from the same group was not significantly affected. Our experiments on the other hand, did not record a reduced endothelium-dependent relaxation in hypertensive compared to normotensive animals, neither in different age groups. Increased norepinephrine-induced contraction occurs even before development of reduced acetylcholine-induced relaxation in SHR rats. We predict that in our experiment hypertension plays a bigger role in the development of endothelial dysfunction than aging (Fig. 2, Ref. 22).

Acute stress responses in salivary alpha-amylase predict increases of plasma norepinephrine

Current biobehavioral research increasingly employs salivary alpha-amylase (sAA) as a surrogate marker for sympathetic nervous system (SNS) activity. While different lines of evidence point to the validity of this assumption, the literature is inconsistent with regard to associations of sAA with well-established SNS indicators, such as plasma norepinephrine (NE) or epinephrine (E). Small samples as well as application of different stress paradigms might be responsible. This study therefore set out to examine the relation between stress-induced sAA activity with NE and E by exposing a larger and less constrained sample to an effective stress protocol. Sixty-six healthy participants (mean age 24.30±4.24 yrs), including n=40 women, n=26 men, n=18 oral contraceptive (OC) users, and n=15 habitual smokers, were recruited and subjected to the Trier Social Stress Test (TSST). Saliva and blood samples were taken at four time points throughout the experiment for later analysis of sAA activity and NE/E concentration, respectively. As expected, sAA, NE, and E showed significant increases in response to the acute stress induction (all p<0.001). Regression analyses (controlling for age, BMI, sex, smoking and OC) revealed that stress responses in sAA significantly predicted stress responses in NE (r=0.326; p=0.025). Interestingly, stress responses in E predicted NE to a lesser extend (β=0.265; p=0.064). E responses showed no association with sAA (β=0.265; p=0.064). Higher sAA levels were found in habitual smokers (F=4.27; p=0.043) and in individuals with lower BMI (F=2.81; p=0.099). In conclusion, current data clearly show an association between stress responses of sAA and plasma NE. This relationship is stronger than the association of norepinephrine and epinephrine responses, thus placing the predictive power of sAA well within the expected range for different SNS markers.

Vasopressin-Mediated Enhancement of Adrenergic Vasoconstriction Involves Both the Tyrosine Kinase and the Protein Kinase C Pathways

Vasopressin is frequently used to treat catecholamine-resistant vasodilatory shock. It enhances the vasoconstrictor effects of catecholamines at concentrations of vasopressin that have none or only minimal intrinsic pressor effects. However, the vascular mechanisms underlying this combined pharmacological approach have not been fully elucidated. We used isometric tension measurements in vascular rings to investigate potential cellular mechanisms. Vascular rings (0.2 mm diameter) were harvested from the superior mesenteric artery of Wistar rats (2 to 4 months of age). Dose-response relationships were derived for vasopressin (VP) and norepinephrine (NE), in the absence and presence of a subpressor dose of VP (10(-9) M). The contribution of tyrosine kinase (TK), the TK pathway proteins SRC and PYK2, as well as protein kinase C (PKC) were determined by measuring the modulating influence of specific inhibitors on the pressor response to NE (10(-5) M) alone and the augmented pressor response to VP (10(-9) M). VP (10(-9) M) had only minimal pressor effect alone (10% of maximal response), but significantly increased the E(max) response to NE (587.8 ± 40.7 vs 331.2 ± 10.4 mg). TK inhibition completely abolished the pressor response to NE (100% vs 1.0% 0.5%) and the augmented VP response alone (100% vs 2.0% ± 1.01%). Both responses were significantly, but equally, decreased by SRC inhibition (63% ± 4.0% and 69% 1.0%). In contrast, inhibition of the TK molecule PYK2 with salicylate had differential inhibitory effects on the vasoconstrictor responses. Salicylate caused a greater inhibition of VP-induced augmented NE response in comparison with NE alone (62.1% ± 7% and 15% ± 2%). Inhibition of either the µ or γ PKC isoform significantly decreased both responses, but the magnitude of the inhibition was significantly different for each isoform. Inhibition of the γ PKC isoform significantly decreased the vasoconstriction responses to both NE and VP plus NE (82.9 ± 3.9 vs 32.8 ± 3.8). Inhibition of the µ PKC isoform significantly decreased both responses and completely abolished the VP-augmented response to NE. These data are consistent with a central role for TK in mediating both the NE response and the VP-augmented response. Moreover, PYK2 and the µ and γ isoforms of PKC seem to play a preferential role in mediating the augmented VP response. The apparent divergent roles of these pathways in mediating NE- versus VP-augmented pressor responses could potentially lead to new targeted therapies in catecholamine-resistant shock.

Hemodynamic and Hormonal Stress Responses to Endotracheal Tube and ProSeal Laryngeal Mask AirwayTMfor Laparoscopic Gastric Banding

The stress responses from tracheal intubation are potentially dangerous in patients with higher cardiovascular risk, such as obese patients. The primary outcome objective of this study was to test whether, in comparison with the endotracheal tube (ETT), the Proseal™ Laryngeal Mask Airway (PLMA™) (Laryngeal Mask Airway Company, Jersey, United Kingdom) reduces blood pressure and norepinephrine responses and the amounts of muscle relaxants needed in obese patients. We assessed hemodynamic and hormonal stress responses, ventilation, and postoperative recovery in 75 morbidly obese patients randomized to receive standardized anesthesia with either an ETT or the PLMA™ for laparoscopic gastric banding. In repeated-measures ANOVA, mean arterial blood pressure and plasma norepinephrine were significantly higher in the ETT group than in the PLMA™ group. In individual pairwise comparisons, blood pressure rose higher in ETT than PLMA™ patients after insertion and removal of airway devices, and after recovery. In ETT compared with PLMA™ patients, plasma norepinephrine was higher after induction of carboperitoneum (mean ± SD, 534 ± 198 and 368 ± 147 and pg/ml, P = 0.001), after airway device removal (578 ± 285 and 329 ± 128 pg/ml, P < 0.0001), and after recovery in postanesthesia care unit (380 ± 167 and 262 ± 95 and pg/ml, P = 0.003). Compared with use of the ETT, the PLMA™ reduced cisatracurium requirement, oxygen desaturation, and time to discharge from both the postanesthesia care unit and the hospital. PLMA™ reduces stress responses and postoperative complaints after laparoscopic gastric banding.

Vildagliptin Reduces Glucagon during Hyperglycemia and Sustains Glucagon Counterregulation during Hypoglycemia in Type 1 Diabetes

The dipeptidyl peptidase-4 inhibitor, vildagliptin, inhibits glucagon secretion at hyperglycemia but appears to enhance glucagon counterregulation during hypoglycemia in type 2 diabetes. The objective of the investigation was to study whether vildagliptin also improves α-cell function in type 1 diabetes (T1D). The study was a single-center, double-blind, randomized, placebo-controlled crossover study involving 28 patients with C-peptide negative and antibody positive T1D [21 males, seven females, glycosylated hemoglobin 57.9 mmol/mol (7.5%)]. Patients received vildagliptin (50 mg twice a day) or placebo as an add-on to their insulin therapy for 4 wk each. On d 28 of the respective treatment period, patients were served a standard meal (500 kcal) to raise the circulating incretin hormone levels followed by a hyperinsulinemic hypoglycemic clamp at 2.5 mmol/liter. The increase in plasma glucagon levels during the 30-min hypoglycemic clamp (min 165-195 of the test) was measured. During the meal, glucagon levels were lower with vildagliptin than with placebo (120 min area under the curve(glucagon) 2.4±0.2 vs. 2.6±0.2 nmol/liter×minutes, P=0.022 for between group difference). In contrast, during hypoglycemia, the glucagon counterregulation was not reduced by vildagliptin (increase in glucagon 1.5±1.0 pmol/liter with vildagliptin vs. 1.7±0.8 pmol/liter with placebo, P=NS). In addition, the counterregulatory responses in epinephrine, norepinephrine, cortisol, and pancreatic polypeptide were not different between the treatments. During the 4-wk treatment period, vildagliptin reduced the mean glycosylated hemoglobin, whereas there was no change with placebo [between group difference was -3.4±1.0 mmol/mol (-0.32±0.09%; P=0.002)] from baseline of 57.9 mmol/mol (7.5%). Vildagliptin, although inhibiting glucagon secretion during hyperglycemia, does not compromise the glucagon counterregulatory response during hypoglycemia in T1D.

Design, synthesis and vasodilative activity of tanshinone IIA derivatives

A new series of tanshinone IIA (DIIA) derivatives were synthesized through the reaction of brominated tanshinone IIA (1-Br DIIA) and aromatic acids in the presence of K2CO3. Twenty compounds were synthesized, and all of them were novel. Vasodilative activities for synthesized compounds were valuated in vitro on the contractile response of vascular thoracic aorta smooth muscle from Wistar rats. The results showed that most compounds exhibited a concentration-dependent inhibition on the contractile response of norepinephrine. Four prepared compounds, 4, 5, 8 and 13 revealed relatively remarkable vasodilative activity.

Neuroendocrine and immune responses to a cognitive stress challenge in veterans with and without PTSD

BackgroundPTSD11Deceased. has been associated with altered hypothalamus–pituitary–adrenal-axis (HPA-axis), immune and sympathetic nervous system (SNS) regulation. The purpose of this study was to evaluate the effect of cognitive stress on these systems in PTSD patients and controls.MethodsThe subjective units of distress score (SUDS), NK-cell response, plasma levels of noradrenalin and ACTH in response to cognitive stress were assessed in male veterans with PTSD (n=15) and age, region and year of deployment matched veterans without psychopathology (n=15).ResultsThe challenge induced an increase in SUDS, noradrenalin, ACTH and NK-cell response in both groups. Baseline levels of ACTH were lower in PTSD patients. The test was experienced as more stressful by PTSD patients and resulted in an augmented ACTH response in patients. The noradrenalin and NK-cell responses showed no group differences. The ACTH response correlated with the severity of symptoms in patients, and the noradrenalin response correlated with the ACTH and NK-cell response in controls, but not in patients.DiscussionPTSD patients experience more distress and present with an exaggerated pituitary response to this stressor. In addition, our results suggest an altered interaction between the HPA-axis, SNS and immune system in PTSD.For the abstract or full text in other languages, please see Supplementary files under Reading Tools online

β2 Adrenoceptor Signaling-induced Muscle Hypertrophy from Blood Flow Restriction: Is There Evidence?

Skeletal muscle hypertrophy and increases in muscular function have been observed following low intensity/load exercise with blood flow restriction (BFR). The mechanisms behind these effects are largely unknown, but have been hypothesized to include a metabolic accumulation induced increase in muscle activation, elevations in growth hormone, and improvements in muscle protein balance. However, many of the aforementioned mechanisms are not present with BFR in the absence of exercise. In these situations, signaling through the β2 adrenoceptor has been hypothesized to possibly contribute to the positive muscle adaptions, possibly in concert with muscle cell swelling. Signaling through the β2 adrenoceptor has been shown to stimulate both muscle protein synthesis and an inhibition of protein degradation through increasing cyclic adenosine monophosphate (cAMP) or signaling via the Gβγ subunit, especially in situations where the basal rates of protein synthesis are already reduced. Every study that has investigated the catecholamine response to BFR in the absence of exercise or in combination with exercise has shown a significant increase above resting conditions. However, from the available evidence, it is unlikely that the norepinephrine response from BFR, particularly with exercise, is playing a prominent role with muscle adaptation in skeletal muscle that is not immobilized by a cast or joint injury.

Oral contraception and energy intake in women: Impact on substrate oxidation during exercise

Oral contraception (OC) and energy intake may play a role in fuel selection during exercise. The aim of this study was to investigate the effect of OCs (OC+ vs. OC-) in fed and fasting conditions on substrate oxidation and metabolic and hormonal responses in women during exercise. Substrate oxidation (respiratory exchange ratio and lipid and carbohydrates oxidation rates), metabolic (glycerol, free fatty acids (FFA), and glucose), and hormonal (insulin, adrenaline, and noradrenaline) responses were determined in 21 women: 10 regularly menstruating women (OC-) and 11 women using OCs (OC+: low-dose monophasic pill; ethinyl estradiol ≤ 30 µg) during 45 min at 65% of maximal oxygen consumption in fasting and postprandial states. At rest, OC+ presented higher low-density lipoprotein cholesterol, total cholesterol, and triglyceride plasma concentrations as compared with OC-. OC status had no influence on substrate oxidation and metabolic and hormonal responses during exercise. In the fasting state, whatever the OC status, women exhibited greater reliance on fat than in postprandial condition. This occurred in the presence of lower plasma insulin concentrations and higher plasma FFA and glycerol levels. The results indicated that the use of low-dose monophasic combined with OCs did not modify fuel selection and metabolic and hormonal responses during exercise in women. The fasting condition, compared with the fed condition, decreased carbohydrate oxidation during exercise, leading to a greater lipid mobilization and utilization whatever the OC status. Thus, in women, the realization of an exercise in either the fed or fasting conditions had a greater impact on substrate oxidation than OC status.

Opioid Receptor Blockade Prevents Exercise-Associated Autonomic Failure in Humans

Hypoglycemia and exercise both induce the release of β-endorphin, which plays an important role in the modulation of the autonomic response during subsequent events. Because opioid receptor (OR) blockade during antecedent hypoglycemia has been shown to prevent hypoglycemia-associated autonomic failure, we hypothesized that OR blockade during exercise would prevent exercise-associated autonomic failure (EAAF). We studied 8 healthy subjects on 2 consecutive days, each of whom participated in three different studies in random order. The protocol on day 1 involved one of the following: 1) two 90-min hyperinsulinemic-euglycemic clamps plus naloxone infusion (control); 2) two 90-min hyperinsulinemic-euglycemic clamps with exercise at 60% Vo2max, plus naloxone infusion (N+); or 3) same protocol as in the N+ group, but with saline infusion only (N−). On day 2, all were studied with stepped hyperinsulinemic-hypoglycemic clamps, using hormone concentrations and glucose turnover as indicators of hypoglycemia counterregulation. Compared with control, N− studies resulted in significantly blunted epinephrine and norepinephrine responses to subsequent hypoglycemia. Conversely, the N+ group exhibited unimpaired hypoglycemia counterregulation, characterized by appropriate increases in epinephrine, norepinephrine, and endogenous glucose production. Thus, OR blockade with naloxone during antecedent exercise prevents the development of acute EAAF by improving the catecholamine responses and by restoring endogenous glucose production.

Plasma Cortisol and noradrenalin concentrations in pigs: automated sampling of freely moving pigs housed in the PigTurn®versus manually sampled and restrained pigs

AbstractMinimising the effects of restraint and human interaction on the endocrine physiology of animals is essential for collection of accurate physiological measurements. Our objective was to compare stress-induced cortisol (CORT) and noradrenalin (NorA) responses in automated vs manual blood sampling in pigs. A total of 16 pigs (30 kg) were assigned to either: (i) automated blood sampling via an indwelling catheter using a novel-penning system called PigTurn®which detects the pig's rotational movement and responds by counter-rotating, allowing free movement while preventing catheter twisting; (ii) automated sampling while exposed to visual and auditory responses of manually sampled pigs; or (iii) manual sampling by jugular venipuncture while pigs were restrained in dorsal recumbency. During sampling of (i), personnel were not permitted in the room; samplings of (ii) and (iii) were performed simultaneously in the same room. Blood samples were collected every 20 min for 120 min and measured for CORT (ng ml−1) using mass spectrometry and NorA (pg ml−1) using High Performance Liquid Chromatography (HPLC). Effects of treatment and time were computed with mixed models adjusted by Tukeypost hoc. CORT and NorA concentrations were lowest in group (i) followed by group (ii), which were not different. However, CORT and NorA levels in manually sampled animals (iii) were highest compared to automated methods (i) and (ii). Plasma concentrations across time were not different for CORT, but NorA concentration at time 0 min was higher than at 120 min. The presence of visual and auditory stimuli evoked by manual sampled animals did not affect non-handled pigs’ responses. Restraint and manual sampling of pigs can be extremely stressful while the automated blood sampling of freely moving pigs, housed in the PigTurn®was significantly less stressful for the animals.

Exclusive interaction between α1D‐adrenergic receptor and pannexin 1 can mediate vasoconstriction

We recently demonstrated that pannexin1 (Panx1) channels release ATP in vascular smooth muscle cells after phenylephrine (PE) stimulation (Billaud et al, Circ Res, 2011). However, the participation of Panx1 in other contractile pathways has not been investigated. Therefore, we stimulated thoracodorsal arteries with angiotensin II, endothelin‐1, serotonin, ATP, KCl, noradrenaline and PE. Only noradrenaline and PE responses were altered by pannexin inhibitors suggesting that these responses occurred through an association between the α1D‐adrenergic receptor (α1D‐AR) and Panx1. These results were verified using duolink imaging of the α1D‐AR/Panx1 interaction. We hypothesized that disruption of this complex would alter vasoconstriction and therefore designed peptides against the Panx1 intracellular loop (IL) and carboxyl tail (CT). Only the Panx1 IL peptide decreased constriction to PE. We further tested the effect of the peptides on the integrity of the Panx1/α1D‐AR complex using co‐immunoprecipitation and verified that the peptides had no effect on Panx1 hexamerization and trafficking. We demonstrated this in vitro by co‐transfection of Panx1 and α1D‐AR plasmids in HEK cells. Electrophysiology confirmed PE‐induced Panx1 activation which was altered by Panx1 IL peptide. Our results show that Panx1 has an exclusive interaction with α1D‐AR in the regulation of vascular tone.

Recurrent spontaneous hypoglycaemia causes loss of neurogenic and neuroglycopaenic signs in infants with congenital hyperinsulinism

Hypoglycaemia-associated autonomic failure (HAAF) with impaired neurogenic and neuroglycopaenic responses occurs in adults following recent, repeated hypoglycaemia. We aimed to evaluate whether HAAF also occurs in patients with infant-onset congenital hyperinsulinism (CHI). A controlled fast was performed in (i) seven CHI infants with initial symptomatic hypoglycaemia and three recent episodes of spontaneous recurrent hypoglycaemia each lasting <5 min and in (ii) seven infants with idiopathic ketotic hypoglycaemia for control. At the time of hypoglycaemia (blood glucose <3 mmol/l or clinical signs), blood was drawn for serum insulin, cortisol, glucagon, adrenalin and nor-adrenalin. Signs of hypoglycaemia were documented. In CHI patients, the ABCC8 and KCNJ11 genes were analysed by denaturing high performance liquid chromatography (DHPLC) and/or direct bidirectional sequencing. Two CHI patients had a paternal ABCC8 mutation, five had no mutations. When repeated hypoglycaemia was provoked, all CHI patients exhibited a complete loss of clinical signs of hypoglycaemia, along with a global blunting of the counter-regulatory hormones cortisol, glucagon, growth hormone, adrenalin and nor-adrenalin responses (median values 256 nmol/l, 23 pmol/l, 5·6 mU/l, 390 pmol/l and 2·9 nmol/l, respectively), irrespective of mutational status. In the controls, hypoglycaemia was always clinically overt with normal counter-regulatory cortisol, glucagon, adrenalin and nor-adrenalin responses (530 nmol/l, 60, 920 pmol/l and 4·0 nmol/l, respectively). Recurrent hyperinsulinaemic hypoglycaemia even of short duration blunts the autonomic, neuroglycopaenic and glucose counter-regulatory hormonal responses in patients with infant-onset CHI resulting in clinically silent hypoglycaemia. Tight, or continuous, glucose monitoring is therefore recommended, especially in conservatively treated patients.

Bilateral thoracoscopic splanchnicectomy for pain in patients with chronic pancreatitis impairs adrenomedullary but not noradrenergic sympathetic function

BackgroundBilateral thoracoscopic splanchnicectomy (BTS) is a well-known technique to alleviate intractable pain in patients with chronic pancreatitis. BTS not only disrupts afferent fibers from the pancreas that mediate pain but also postganglionic sympathetic fibers, which originate in segments T5–T12 and which innervate the vasculature of the liver, pancreas, and the adrenal gland. The purpose of this study was to assess whether and how BTS affects sympathetic noradrenergic and adrenomedullary function in patients with chronic pancreatitis.MethodsSixteen patients with chronic pancreatitis for at least 1 year underwent autonomic function testing before and 6 weeks after BTS for intractable pain. Testing was performed during supine rest and during sympathetic stimulation when standing.ResultsSupine and standing systolic and diastolic blood pressure were significantly lower post-BTS compared with pre-BTS (P = 0.001). One patient showed orthostatic hypotension after BTS. Baseline plasma norepinephrine levels and plasma norepinephrine responses to sympathetic activation during standing were not reduced by BTS. In contrast, supine plasma epinephrine levels and responses during standing were significantly reduced (P < 0.001). Parasympathetic activity was unaffected by BTS as shown by unaltered Valsalva ratio, I-E difference, and ΔHRmax.ConclusionsBTS for pain relief in patients with chronic pancreatitis reduced adrenomedullary function, due to disruption of the efferent sympathetic fibers to the adrenal gland. BTS did not affect noradrenergic sympathetic activity, although blood pressure was lower after the sympathectomy.

Desipramine selectively potentiates norepinephrine-elicited ERK1/2 activation through the α2A adrenergic receptor

The precise physiological effects of antidepressant drugs, and in particular their actions at non-monoamine transporter targets, are largely unknown. We have recently identified the tricyclic antidepressant drug desipramine (DMI) as a direct ligand at the α2A adrenergic receptor (AR) without itself driving heterotrimeric G protein/downstream effector activation [5]. In this study, we report our novel finding that DMI modulates α2AAR signaling in response to the endogenous agonist norepinephrine (NE). DMI acted as a signaling potentiator, selectively enhancing NE-induced α2AAR-mediated ERK1/2 MAPK signaling. This potentiation of ERK1/2 activation was observed as an increase in NE response sensitivity and a prolongation of the activation kinetics. DMI in a physiologically relevant ratio with NE effectively turned on ERK1/2 signaling that is lacking in response to physiological NE alone. Further, the DMI-induced ERK1/2 potentiation relied on heterotrimeric Gi/o proteins and was arrestin-independent. This modulatory effect of DMI on NE signaling provides novel insight into the effects of this antidepressant drug on the noradrenergic system which it regulates, insight which enhances our understanding of the therapeutic mechanism for DMI.

Vascular BK Channel Deficiency Exacerbates Organ Damage and Mortality in Endotoxemic Mice

We determined the contribution of vascular BK channels to endotoxin (lipopolysaccharide, LPS)-induced hypotension, organ damage, and mortality using smooth muscle BK channel deficiency (BK channel β1-subunit knockout, BK β1-KO) mice. BK β1-KO mice were more sensitive to LPS-induced mortality compared with wild-type mice. After LPS (20 mg/kg, intraperitoneally), BK β1-KO mice had a more rapid fall in heart rate and blood pressure (measured by radiotelemetry), shorter latency to mortality, and higher mortality rate than wild-type mice. Twenty-two hours after LPS treatment, wild-type and BK β1-KO mice had reduced norepinephrine reactivity and impaired constrictor responses to the BK channel blocker paxilline in mesenteric arteries in vitro and higher iNOS expression in the heart, but not in mesenteric arteries. Endotoxemic BK β1-KO mice also showed more severe lung and intestinal injury, higher myeloperoxidase activity and polymorphonuclear neutrophil infiltration in lung and liver. Endotoxemic BK β1-KO mice had higher plasma tumor necrosis factor α and interleukin 6 levels at 22 hours, but not 6 hours post-LPS. Exaggerated mortality in BK β1-KO mice also occurred in the cecal ligation/puncture model of septic shock. Reduced vascular BK channel function does not protect against hypotension in the early stage of septic shock; in the later stage, smooth muscle BK channel deficiency enhances organ damage and mortality.

Endocrine response to acute stress in pigs with differing backtest scores

In an attempt to quantify a pig's behavioral reaction to stress, pigs in previous studies were subjected to a backtest. Pigs scoring high on the backtest were thought to have a greater reaction to stress than those scoring lower on the test. To further understand this reaction eighty-eight gilts from 19L were tested twice using the backtest. Gilts with the greatest (n=15, High) and least (n=15, Low) total time struggling were then selected for acute stress challenges. Pigs, in two replicates, were fitted with jugular cannulas at approximately 150d of age. An acclimation time of 24h was given between cannulation, testing and between tests. Blood samples were collected at 10 time points (−60, −30, 0, 5, 10, 15, 30, 60, 90, and 120m relative to the application of the stressor). Pigs were challenged with 0.11mol/kg BW of corticotropin releasing hormone (CRH) (n=9 High, n=9 Low) injected through the cannula to test the proposed model. The first stressor was snaring the pig with a nose-snare for 1min (n=10 High, n=9 Low). The second stressor was depriving the pigs of feed for 24h followed by placing feed next to the pig but out of reach (n=6 High, n=7 Low). Plasma samples were assayed for cortisol, epinephrine, and norepinephrine. Data were analyzed using a model including fixed effects of time, replicate, and group (High or Low) and all two-way interactions. In response to the CRH challenge, cortisol levels peaked at 10m and returned to basal levels by 90m. Least squares means response in norepinephrine of the High exceeded Low at 5m by 376±99pg/mL. Cortisol, epinephrine, and norepinephrine response to snaring was similar. Cortisol peaked at 10m and epinephrine and norepinephrine at 5m. The response to feed deprivation failed to exceed basal levels. Response to the CRH challenge demonstrated the appropriateness of the model and that High and Low pigs were equally capable of responding. The biological significance of differences between High and Low groups in norepinephrine levels at 5m following snaring is unknown.

Busulphan-Cyclophosphamide Cause Endothelial Injury, Remodeling of Resistance Arteries and Enhanced Expression of Endothelial Nitric Oxide Synthase

Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late effects on vascular system.