Induced Catecholamine Release Research Articles

Noradrenergic modulation of stress induced catecholamine release: Opposing influence of FG7142 and yohimbine.

Life stress modulates decision making, particularly in the face of risk, in some cases prompting vulnerable populations to make suboptimal, life-altering choices. In the brain, stress is known to alter the extracellular release of catecholamines in structures such as basolateral amygdala (BLA) and nucleus accumbens (NAc), but the relationship between catecholamines and decision-making behavior under stress has not been systemically explored. We developed an operant touchscreen decision-making task for rats comprising elements of loss aversion and risk seeking behavior. Rats were first injected systemically with an adrenergic -receptor agonist (guanfacine) and antagonist (yohimbine), as well as a partial inverse GABAA agonist, FG 7142, known to induce anxiety and stress related physiological responses in a variety of species, including humans. We then used fiber photometry to monitor NE in the basolateral amygdala (BLA), and DA activity in the nucleus accumbens (NAc) while animals engaged in decision-making and following systemic injections of FG 7142 and yohimbine. Neither yohimbine nor guanfacine had any impact on decision making strategy but altered motivational state with yohimbine making the animal almost insensitive to the reward outcome. The pharmacological induction of stress with FG 7142 biased the rats' decisions towards safety, but this bias shifted toward risk when co-treated with yohimbine. In the BLA and NAc, the FG 7142 altered catecholamine release, with systemic yohimbine producing opposing effects on NE and DA release. Stress induced changes in catecholamine release in the BLA and NAc can directly influence loss sensitivity, decisions and motivation, which can be modulated by the adrenoreceptor antagonist, yohimbine.

Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria.

6-Nitrodopamine (6-ND) is released from rat isolated atria being 100 times more potent than noradrenaline and adrenaline, and 10,000 times more potent than dopamine as a positive chronotropic agent. The present study aimed to investigate the interactions of 6-ND with the classical catecholamines, phosphodiesterase (PDE)-3 and PDE4, and the protein kinase A in rat isolated atria. Atrial incubation with 1 pM of dopamine, noradrenaline, or adrenaline had no effect on atrial frequency. Similar results were observed when the atria were incubated with 0.01 pM of 6-ND. However, co-incubation of 6-ND (0.01 pM) with dopamine, noradrenaline, or adrenaline (1 pM each) resulted in significant increases in atrial rate, which persisted over 30 min after washout of the agonists. The increased atrial frequency induced by co-incubation of 6-ND with the catecholamines was significantly reduced by the voltage-gated sodium channel blocker tetrodotoxin (1 µM, 30 min), indicating that the positive chronotropic effect of 6-ND is due in part to activation of nerve terminals. Pre-treatment of the animals with reserpine had no effect on the positive chronotropic effect induced by dopamine, noradrenaline, or adrenaline; however, reserpine markedly reduced the 6-ND (1 pM)-induced positive chronotropic effect. Incubation of the rat isolated atria with the protein kinase A inhibitor H-89 (1 µM, 30 min) abolished the increased atrial frequency induced by dopamine, noradrenaline, and adrenaline, but only attenuated the increases induced by 6-ND. 6-ND induces catecholamine release from adrenergic terminals and increases atrial frequency independently of PKA activation.

Night Transplantation and Catecholamine Exposure Accelerates In Vivo Leukemia Development By Enhancing Bone Marrow Homing Capacity in Leukemic Cells

Xenografts in immune suppressed mice are routinely used for in vivostudies on human acute myeloid leukemia (AML). However, a significant proportion of primary AML samples (»50-60% of cases) were engrafting in mice with particularly latency. Here, we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) accelerates the engraftment of human AML cells in NOD/SCID/IL2Rgnull(NSG) mice. Equal numbers of freshly thawed AML cells from the same donor (n=5 patients) were transplanted into sublethally irradiated gender-matched 6 to 8 weeks old mice at day or at night (n=3-5 mice per condition and patient). Transplanted mice were monitored for leukemic engraftment in peripheral blood (PB), bone marrow (BM) and other hematopoietic organs using flow cytometry and immunohistochemistry. Enhanced leukemic engraftment was observed in night vs. day transplanted mice for each AML case (Fig. A). Interestingly, in 2/5 samples, engraftment was exclusively detected with the night transplant condition. Limiting dilutions showed that night transplantation allows leukemia (Fig. B) induction from lower cell numbers. Results were confirmed in a syngeneic MLL-PTD/FLT3-ITD mouse leukemia model (Fig. C): mice analyzed at the same time-point after night versus day transplantation showed enhanced splenomegaly (401.2 mg vs. 234.5 mg, p<0.05, Fig. D) and abnormal blood counts (white blood cells numbers 55.41x109vs. 18.63x109, p<0.01). To explore underlying mechanisms, homing assays were performed with fluorescently labelled human AML cells injected via tail vein and analyzed 12 hours later. Significantly higher BM colonization was found with cells injected at night versus day times (0.31±0.09 vs. 0.13±0.18, p<0.001, n=7 AML patients, Fig. E). Intriguingly, mice transplanted at night showed stress-related behavioral abnormalities. We thus hypothesized that night transplantation induced catecholamine release may affect homing and leukemogenesis. ELISAs indeed revealed enhanced epinephrine levels in PB (318.00±12.96 vs. 122.20±47.11 ng/mL, p<0.001) and BM (21.08±1.04 vs. 13.50±3.57 ng/ml, p<0.05) of night vs. day transplanted animals. Adrenergic receptors have been previously described to express in BM cells, and to regulate hematopoietic stem and progenitor cells (HSPCs) mobilization, Here, we show that these pathways are also important during leukemogenesis. Blocking adrenergic signaling with the b1/2-blocker propranolol prior to night transplantation (10mg/kg, intraperitoneal (i.p.) injection, 1h pre-transplant) reduced homing (0.22±0.17 vs. 0.39±0.22% leukemic cells, p=0.09) and long-term engraftment (23±7.42 vs. 80.2±11.1% leukemic blasts in BM, p<0.0001), and enhanced survival (Fig. F) in NSG mice transplanted with human AML cells and in syngeneic murine leukemia models. Consistently, treatment of mice with epinephrine prior to transplantation (2mg/kg, i.p., 1h pre-transplant) enhanced homing (0.38±0.09 vs. 0.16±0.04%, p<0.0001) and accelerated long-term leukemia induction (12.49±5.23 vs. 4.43±3.6%, p<0.01).. Surprisingly, even though all our AML samples express the homing molecule CXCR4, blocking CXCR4 (anti-CXCR4 antibody, 50mM) prior to transplantation could not counteract epinephrine-driven homing (0.032±0.017 vs. 0.038±0.025% leukemic cells in BM, p=0.53), indicating that the stimulating effect of adrenergic signaling on homing was independent of the SDF1-CXCR4 axis. Notably, transplantations of healthy human cord-blood CD34+ HSPCs or of mouse BM Lin- cells revealed no difference in the hematopoietic reconstitution in night vs. day transplantations (Fig. G). This indicates that the observed mechanisms are leukemia specific. To explore further potential mechanisms, RNAseq was performed on MS-5 cells, a mouse stromal cell line, treated with either epinephrine or vehicle control. Preliminary analyses indicate upregulated vitamin D receptor (VDR) expression (log2FC=1.18, p=1.32x1012) as potentially involved in catecholamine-induced homing of leukemic cells. We conclude that enhanced catecholamine levels (e.g. induced by night transplantation) accelerate leukemia development from AML cells, by enhancing the homing of leukemic cells to BM niches via SDF1-CXCRR4 independent, leukemic specific mechanisms. Figure Disclosures No relevant conflicts of interest to declare.

Cardiogenic shock secondary to phaeochromocytoma: adrenalectomy on multiple mechanical circulatory support devices

Introduction Phaechromocytoma is a neuroendocrine tumour of the chromaffin cells of the adrenal medulla. Tumours secrete catecholamines and typically present with hypertensive crisis or, in rare circumstances, cardiogenic shock. Case Presentation A 33 year old male presented with hypertensive crisis and fulminant cardiogenic shock. He required mechanical ventilation for hypoxaemia secondary to acute pulmonary oedema. A CT aortogram was performed for presumed aortic dissection which revealed a necrotic, enhancing adrenal mass. Due to severe left ventricular dysfunction, he was referred to our hospital for mechanical circulatory support (MCS). Peripheral veno-arterial extracorporeal membrane oxygenation (VA-ECMO) was established in the referral hospital and he was transferred to our intensive care unit. An Impella-CP was then inserted to offload an increasingly dilated left ventricle. Hypertensive crises continued to occur despite maximum medical therapy, and it was determined the only viable option to control these paroxysms of hypertension was to perform adrenalectomy. Surgery was successfully performed whilst on dual MCS devices (VA-ECMO and Impella). Preoperatively the patient had intravenous alpha and beta adrenoceptor blockade and fluid status was optimised to address the chronic circulating volume depletion. Glucose and electrolyte imbalances were corrected. Intra-operative management focused on blood pressure control, heart rate and arrhythmia control. Agents that induce catecholamine release were avoided and tumour manipulation was minimised. Hypotension after tumour removal was anticipated and developed relatively quickly requiring infusions of noradrenaline, adrenaline and vasopressin to support an adequate arterial pressure. His post-operative recovery was initially favourable and myocardial function had improved to the point of planning ECMO explanation. Despite cardiovascular stability the patient suffered a catastrophic intracranial haemorrhage and treatment was withdrawn. Discussion Cardiogenic shock secondary to phaeochromoctyoma can be difficult to manage and has high mortality. The evolution of short to medium term MCS devices has provided clinicians with additional therapeutic options to manage refractory cases of this uncommon cause of cardiogenic shock. The authors hope this case will add to an expanding library of practice of using MCS in phaeochromocytoma.

Catecholamine Exposure Accelerates In Vivo Leukemogenesis in Acute Myeloid Leukemia Patient Derived Xenografts

Mouse xenografts are routinely used for in vivo studies on human acute myeloid leukemia (AML) cells. However, a significant proportion of primary AML samples (~50-60% of cases) are not able to repopulate mice, or respectively require a particularly long time (up to 9 months) to show detectable engraftment in such models. Here we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) strongly improved the engraftment of human AML cells in NOD/SCID/IL2Rgnull (NSG) mice. Sublethally irradiated gender-matched NSG mice of 6-8 weeks of age (n=40) were transplanted with primary AML cells (n=5 patients). For each sample, equal numbers of freshly thawed AML cells were injected via the tail vein at both time-points; transplanted mice were afterwards monitored for human leukemic engraftment in peripheral blood (PB) and bone marrow (BM) using multicolor flow cytometry and immunohistochemistry. In all n=5 analyzed AML patient samples, transplantation at night strongly promoted leukemogenesis, with 2/5 samples showing engraftment only in the night transplant condition and 3/5 samples showing engraftment in both but significantly higher leukemic burden in night vs. day transplanted mice (Fig. 1a), although similar leukemia-specific mutations were retrieved in mice of the two conditions by next generation sequencing analysis (Fig. 1b). Limiting dilution experiments (n=2 AML samples) further confirmed these findings and showed that in fact lower numbers of AML cells were required to initiate leukemia when cells were transplanted at night versus day times.Given that the immediate process that happens after intravenous injection of AML cells into mice involves homing to BM niches, we hypothesized that the time-point of transplantation influences engraftment by modulating homing. Therefore, we performed homing assays as described for healthy hematopoietic stem/progenitor cells (HSPCs); we injected equally treated human CFSE labeled AML cells via tail vein injection at day versus night time points and analyzed murine BMs 8 hours later for the presence of human leukemic cells. Indeed, significantly higher BM colonization by leukemic cells was observed after night vs. day time injections (0.31±0.09 vs. 0.13±0.18, p<0.001, n= 7 AML patient samples).Since mice transplanted at night showed stress-related behavioral abnormalities, we hypothesized that this transplantation method might induce catecholamine release. ELISAs indeed revealed enhanced epinephrine levels in PB (318.00±12.96 vs. 122.20±47.11 ng/mL, p<0.001) as well as BM (21.08±1.04 vs. 13.50±3.57 ng/mL, p<0.05) of night vs. day transplanted animals. The role of catecholamines influencing leukemia initiation is underinvestigated; b-adrenergic receptors are expressed on several cell types in the BM including HSCs, osteoblasts and mesenchymal stem cells and catecholamine release was shown to regulate HSC egress from BM niches. We hypothesized that enhanced adrenergic signaling may promote leukemogenesis by enhancing leukemic cell homing to BM niches, which in this setting are more readily available due to enhanced healthy HSC egress. To explore this hypothesis, we treated mice with the b1/2-blocker propranolol prior to night transplantation (10mg/kg, intraperitoneal (i.p) injection, 1h pre-transplant, to reverse the “night effect”) and respectively with epinephrine prior to day transplantation (2mg/kg, i.p injection, 1h pre-transplant, to mimic the “night effect”). Indeed, propranolol treatment reduced homing and delayed leukemogenesis in night transplanted mice. Vice-versa, epinephrine enhanced AML cell homing and accelerated long-term leukemia induction from patient derived AML cells and cells of the EOL-1 cell line, and these effects could be counteracted by co-treatment with propranolol. Interestingly, in vitro co-cultures with epinephrine versus empty carrier control enhanced AML cell but not healthy HSPC adhesion to MS5 stroma cells (shown for n=3 cell lines and n=8 primary AML), suggesting that enhanced catecholamine levels may specifically induce AML cell adhesion.We conclude that enhanced catecholamine levels can accelerate leukemia development from AML cells transplanted in xenograft models by facilitating BM niche colonization by AML cells. Catecholamines might in general promote leukemia progression by favoring the retention of leukemic versus healthy HSCs in BM niches. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reverse Takotsubo Cardiomyopathy Associated With the Consumption of an Energy Drink

A 24-year-old man presented to the emergency room with chest pain, acute respiratory failure, and palpitations shortly after the ingestion of small amounts of an energy drink in little cups one after another. He was afebrile, with frequent runs of supraventricular and ventricular tachycardia and underlying sinus tachycardia. The patient was treated with fluids and metoprolol but required intubation because of progressive hypoxia. Chest x-ray revealed bilateral fluffy pulmonary infiltrates. His ECG was free of ischemic changes, and an echocardiogram at that time showed hypokinesis of all basal left ventricular segments with apical sparing and an ejection fraction of 35% (Figure 1 and Movie I in the online-only Data Supplement). His troponin was mildly increased and serially measured brain natriuretic peptide was elevated at 8000 pg/mL. Figure 1. ECG recorded at admission showing sinus tachycardia and nonspecific T-wave inversion in leads I and aVL. After treatment with furosemide, nitroglycerine, heparin, and aspirin, the patient responded with clinical improvement of vital signs and chest x-ray. Episodes of agitation and delirium ensued, delaying extubation. Computerized tomography brain scan and electroencephalogram were negative, and toxicological testing demonstrated no cocaine, cannabis, or …

An Unsuspected Property of Natriuretic Peptides

Background— Although natriuretic peptides are considered cardioprotective, clinical heart failure trials with recombinant brain natriuretic peptide (nesiritide) failed to prove it. Unsuspected proadrenergic effects might oppose the anticipated benefits of natriuretic peptides. Methods and Results— We investigated whether natriuretic peptides induce catecholamine release in isolated hearts, sympathetic nerve endings (cardiac synaptosomes), and PC12 cells bearing a sympathetic neuron phenotype. Perfusion of isolated guinea pig hearts with brain natriuretic peptide elicited a 3-fold increase in norepinephrine release, which doubled in ischemia/reperfusion conditions. Brain natriuretic peptide and atrial natriuretic peptide also released norepinephrine from cardiac synaptosomes and dopamine from nerve growth factor–differentiated PC12 cells in a concentration-dependent manner. These catecholamine-releasing effects were associated with an increase in intracellular calcium and abolished by blockade of calcium channels and calcium transients, demonstrating a calcium-dependent exocytotic process. Activation of the guanylyl cyclase-cyclic GMP-protein-kinase-G system with nitroprusside or membrane-permeant cyclic GMP analogs mimicked the proexocytotic effect of natriuretic peptides, an action associated with an increase in intracellular cyclic AMP (cAMP) and protein-kinase-A activity. Cyclic AMP enhancement resulted from an inhibition of phosphodiesterase type 3–induced cAMP hydrolysis. Collectively, these findings indicate that, by inhibiting phosphodiesterase type 3, natriuretic peptides sequentially enhance intracellular cAMP levels, protein kinase A activity, intracellular calcium, and catecholamine exocytosis. Conclusions— Our results show that natriuretic peptides, at concentrations likely to be reached at cardiac sympathetic nerve endings in advanced congestive heart failure, promote norepinephrine release via a protein kinase G–induced inhibition of phosphodiesterase type 3–mediated cAMP hydrolysis. We propose that this proadrenergic action may counteract the beneficial cardiac and hemodynamic effects of natriuretic peptides and thus explain the ineffectiveness of nesiritide as a cardiac failure medication.

Intensive Insulin Therapy after Decompression Surgery for Severe Traumatic Brain Injury

Following acute traumatic brain injury, stress induced catecholamine release can result in elevated blood glucose levels. Hyperglycemia has been considered a stress response and a simple reflection of the injury severity. In animal studies of ischemic and hemorrhagic stroke, hyperglycemia has been associated with increased ischemic brain damage, edema, and peri-lesional cell death. Early hyperglycemia in rats with traumatic brain injury (TBI) was associated with a larger contusion area and increased inflammation. Current guidelines for acute ischemic stroke suggest that interventions are indicated if a glucose level is greater than 180 mg/dL. Since a landmark trial which demonstrated a significant decrease in mortality using intensive insulin protocol targeting blood glucose levels between 80 and 110 mg/dL in surgical intensive care patients, tight glucose control has been widely adopted in the critical setting. Traditionally, the authors have collaborated with endocrinologists for the control of hyperglycemia in neurosurgical intensive care unit (NSICU), especially in cases with more than 200 mg/dL of blood glucose level. The different treatments affect the clinical outcomes of patients with severe TBI. Therefore, we focus on the craniectomy, a uniform surgical intervention that is widely performed and useful for the treatment of TBI. Here, we have evaluated the feasibility of intensive insulin therapy for patients with severe TBI who underwent decompressive craniectomy. Received: February 14, 2012 / Revised: March 9, 2012 Accepted: March 10, 2012 Address for correspondence: Seung-Ho Yang, MD Department of Neurosurgery, St. Vincent’s Hospital, College of Medicine, The Catholic University of Korea, 93 Jungbu-daero, Paldal-gu, Suwon 442-723, Korea Tel: +82-31-249-8304, Fax: +82-31-245-5208 E-mail: 72ysh@catholic.ac.kr

Multi-target novel neuroprotective compound ITH33/IQM9.21 inhibits calcium entry, calcium signals and exocytosis

Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca2+ entry and neuronal Ca2+ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K+-evoked [Ca2+]c transients with an IC50 of 5.31μM. At 10μM, the compound decreased the amplitude and area of the Ca2+ transient elicited by challenging single fura-2-loaded cells with high K+, by 40% and 80%, respectively. This concentration also caused a blockade of K+-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca2+ currents (IC50=6.52μM). At 10μM, ITH/IQM also inhibited the Ca2+-dependent outward K+ current, leaving untouched the voltage-dependent component of IK. The inward Na+ current was unaffected. Inhibition of depolarisation-elicited Ca2+ entry, [Ca2+]c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia.

Calcium signalling mediated through α7 and non-α7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release.

Ca(2+) signalling and exocytosis mediated by nicotinic receptor (nAChR) subtypes, especially the α7 nAChR, in bovine chromaffin cells are still matters of debate. We have used chromaffin cell cultures loaded with Fluo-4 or transfected with aequorins directed to the cytosol or mitochondria, several nAChR agonists (nicotine, 5-iodo-A-85380, PNU282987 and choline), and the α7 nAChR allosteric modulator PNU120596. Minimal [Ca(2+) ](c) transients, induced by low concentrations of selective α7 nAChR agonists and nicotine, were markedly increased by the α7 nAChR allosteric modulator PNU120596. These potentiated responses were completely blocked by the α7 nAChR antagonist α-bungarotoxin (α7-modulated-response). Conversely, high concentrations of the α7 nAChR agonists, nicotine or 5-iodo-A-85380 induced larger [Ca(2+) ](c) transients, that were blocked by mecamylamine but were unaffected by α-bungarotoxin (non-α7 response). [Ca(2+) ](c) increases mediated by α7 nAChR were related to Ca(2+) entry through non-L-type Ca(2+) channels, whereas non-α7 nAChR-mediated signals were related to L-type Ca(2+) channels; Ca(2+) -induced Ca(2+) -release contributed to both responses. Mitochondrial involvement in the control of [Ca(2+) ](c) transients, mediated by either receptor, was minimal. Catecholamine release coupled to α7 nAChRs was more efficient in terms of catecholamine released/[Ca(2+) ](c) . [Ca(2+) ](c) and catecholamine release mediated by α7 nAChRs required an allosteric modulator and low doses of the agonist. At higher agonist concentrations, the α7 nAChR response was lost and the non-α7 nAChRs were activated. Catecholamine release might therefore be regulated by different nAChR subtypes, depending on agonist concentrations and the presence of allosteric modulators of α7 nAChRs.

Trace amines depress D2‐autoreceptor‐mediated responses on midbrain dopaminergic cells

Although trace amines (TAs) are historically considered 'false neurotransmitters' on the basis of their ability to induce catecholamine release, there is evidence that they directly affect neuronal activity via TA receptors, ligand-gated receptor channels and/or sigma receptors. Here, we have investigated the effects of two TAs, tyramine (TYR) and beta-phenylethylamine (beta-PEA), on electrophysiological responses of substantia nigra pars compacta (SNpc) dopaminergic cells to the D(2) receptor agonist, quinpirole. Electrophysiological recordings of D(2) receptor-activated G-protein-gated inward rectifier K(+) channel (GIRK) currents were performed on dopaminergic cells from midbrain slices of mice and on Xenopus oocytes expressing D(2) receptors and GIRK channels. TYR and beta-PEA reversibly reduced D(2) receptor-activated GIRK currents in a concentration-dependent manner on SNpc neurones. The inhibitory effect of TAs was still present in transgenic mice with genetically deleted TA(1) receptors and they could not be reproduced by the selective TA(1) agonist, o-phenyl-3-iodotyramine (O-PIT). Pretreatment with antagonists of sigma1 and sigma2 receptors did not block TA-induced effects. In GTPgammaS-loaded neurones, the irreversibly-activated GIRK-current was still reversibly reduced by beta-PEA. Moreover, beta-PEA did not affect basal or dopamine-evoked GIRK-currents in Xenopus oocytes. TAs reduced dopamine-induced responses on SNpc neurones by acting at sites different from TA(1), sigma-receptors, D(2) receptors or GIRK channels. Although their precise mechanism of action remains to be identified, TAs, by antagonizing the inhibitory effects of dopamine, may render dopaminergic neurones less sensitive to autoreceptor feedback inhibition and hence enhance their sensitivity to stimulation.

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

1. The Na(+)/Ca(2+) exchanger (NCX) exchanges Na+ and Ca(2+) bidirectionally through the forward mode (Ca(2+) extrusion) or the reverse mode (Ca(2+) influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na(+)-loaded cells were prepared by treatment with 100 micromol/L ouabain and 50 micromol/L veratridine. Incubation of Na(+)-loaded cells with Na(+)-free solution in the presence of the Ca(2+) channel blockers nicardipine (3 micromol/L) and omega-conotoxin MVIIC (0.3 micromol/L) caused Ca(2+) uptake and catecholamine release. 2. The Na(+)-dependent Ca(2+) uptake and catecholamine release were inhibited by 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400; 1 micromol/L) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]isothiourea (KB-R7943; 10 micromol/L), both NCX inhibitors. These results indicate that the Na(+)-dependent responses are mostly due to activation of the NCX working in the reverse mode. 3. In addition, we examined the effects of PKC inhibitors and an activator on the NCX-mediated Ca(2+) uptake and catecholamine release. Bisindolylmaleimide I (0.3-10 micromol/L) and chelerythrine (3-100 micromol/L), both PKC inhibitors, inhibited NCX-mediated responses. In contrast, phorbol 12,13-dibutyrate (0.1-10 micromol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na(+)-dependent catecholamine release, had a little or no effect on high K(+)-induced catecholamine release in intact cells or on Ca(2+)-induced catecholamine release in beta-escin-permeabilized cells. 4. These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

Regulation of catecholamine release and tyrosine hydroxylase in human adrenal chromaffin cells by interleukin‐1β: role of neuropeptide Y and nitric oxide

Adrenal chromaffin cells synthesize and secrete catecholamines and neuropeptides that may regulate hormonal and paracrine signaling in stress and also during inflammation. The aim of our work was to study the role of the cytokine interleukin-1beta (IL-1beta) on catecholamine release and synthesis from primary cell cultures of human adrenal chromaffin cells. The effect of IL-1beta on neuropeptide Y (NPY) release and the intracellular pathways involved in catecholamine release evoked by IL-1beta and NPY were also investigated. We observed that IL-1beta increases the release of NPY, norepinephrine (NE), and epinephrine (EP) from human chromaffin cells. Moreover, the immunoneutralization of released NPY inhibits catecholamine release evoked by IL-1beta. Moreover, IL-1beta regulates catecholamine synthesis as the inhibition of tyrosine hydroxylase decreases IL-1beta-evoked catecholamine release and the cytokine induces tyrosine hydroxylase Ser40 phosphorylation. Moreover, IL-1beta induces catecholamine release by a mitogen-activated protein kinase (MAPK)-dependent mechanism, and by nitric oxide synthase activation. Furthermore, MAPK, protein kinase C (PKC), protein kinase A (PKA), and nitric oxide (NO) production are involved in catecholamine release evoked by NPY. Using human chromaffin cells, our data suggest that IL-1beta, NPY, and nitric oxide (NO) may contribute to a regulatory loop between the immune and the adrenal systems, and this is relevant in pathological conditions such as infection, trauma, stress, or in hypertension.

Brief Communication: Radiographic Contrast Infusion and Catecholamine Release in Patients With Pheochromocytoma

Contrast-enhanced computed tomography (CT) is useful for localizing pheochromocytoma. However, in patients with suspected pheochromocytoma, CT is often canceled or not performed because of the strong belief that intravenous contrast may induce hypertensive crisis. To examine whether intravenous low-osmolar contrast administration during CT induces catecholamine release that increases blood pressure or heart rate. Prospective study. Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland. 22 patients with pheochromocytoma (15 nonadrenal and 7 adrenal) and 8 unmatched control participants without pheochromocytoma. Plasma catecholamine levels, blood pressure, and heart rate. Plasma catecholamine levels within and between groups did not significantly differ before and after intravenous administration of low-osmolar CT contrast. Patients with pheochromocytoma experienced a clinically and statistically significant increase in diastolic blood pressure that was not accompanied by corresponding increases in plasma catecholamine levels. The difference became non-statistically significant after adjustment for use of alpha- and beta-blockers. The study lacked a placebo group, and the sample was relatively small. Intravenous low-osmolar contrast-enhanced CT can safely be used in patients with pheochromocytoma who are not receiving alpha- or beta-blockers. Eunice Kennedy Shriver National Institute of Child Health and Development, National Institutes of Health.

Reinvestigation of the effect of orexin A on catecholamine release from adrenal chromaffin cells

Orexins have been shown to be implicated in the regulation of adrenal medulla functions. However, there are still inconsistent investigations on the effects of orexins on catecholamine release from chromaffin cells in varying species. In the present study, using the carbon-fiber amperometry, we investigated whether orexin A would stimulate catecholamine release from rat and mouse adrenal chromffin cells. Puff application of orexin A dose-dependently induced amperometric currents in the cultured rat chromaffin cells, which was completely blocked by the selective OX1R antagonist SB-334867 or by the removal of extracellular calcium. Likewise, in the mouse adrenal medulla slices, orexin A also induced catecholamine release mainly through the activation of OX1R. These results gain insight into our understanding of the pharmacological relevance of orexin system in modulating neuroendocrine functions.

The role of hyperventilation: hypocapnia in the pathomechanism of panic disorder

The authors present a profile of panic disorder based on and generalized from the effects of acute and chronic hyperventilation that are characteristic of the respiratory panic disorder subtype. The review presented attempts to integrate three premises: hyperventilation is a physiological response to hypercapnia; hyperventilation can induce panic attacks; chronic hyperventilation is a protective mechanism against panic attacks. A selective review of the literature was made using the Medline database. Reports of the interrelationships among panic disorder, hyperventilation, acidosis, and alkalosis, as well as catecholamine release and sensitivity, were selected. The findings were structured into an integrated model. The panic attacks experienced by individuals with panic disorder develop on the basis of metabolic acidosis, which is a compensatory response to chronic hyperventilation. The attacks are triggered by a sudden increase in (pCO2) when the latent (metabolic) acidosis manifests as hypercapnic acidosis. The acidotic condition induces catecholamine release. Sympathicotonia cannot arise during the hypercapnic phase, since low pH decreases catecholamine sensitivity. Catecholamines can provoke panic when hyperventilation causes the hypercapnia to switch to hypocapnic alkalosis (overcompensation) and catecholamine sensitivity begins to increase. Therapeutic approaches should address long-term regulation of the respiratory pattern and elimination of metabolic acidosis.

Catecholamine outflow from mouse and rat brain slice preparations evoked by nicotinic acetylcholine receptor activation and electrical field stimulation

Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (beta2 and alpha5) knockout mice for a comparison with rat brain slice preparations. Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists. When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations. Targeted deletion of the beta2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions.

Neuropeptide Y regulates catecholamine release evoked by interleukin-1β in mouse chromaffin cells

Activation of the hypothalamic-pituitary–adrenal gland (HPA) axis can modulate the immune system. Cytokines and neuropeptide Y (NPY) are potent regulators of the HPA axis and are both produced by the adrenal medulla. The cytokine interleukin-1β (IL-1β) belongs to the interleukin-1 family along with interleukin-1α and the interleukin receptor antagonist (IL-1ra). The aim of the present study was to determine the interaction between NPY and IL-1β in catecholamine (norepinephrine, NE and epinephrine, EP) release from mouse chromaffin cells in culture. We found that IL-1β increased the constitutive release of NPY, NE and EP from mouse chromaffin cells. This IL-1β stimulatory effect was blocked by IL-1ra. The immunoneutralization of NPY and the use of the NPY Y 1 receptor antagonist (BIBP 3226) inhibited the stimulatory effect of IL-1β on catecholamine release from these cells. The present work shows that IL-1β induces catecholamine release, and in turn this peptide will induce an additional increase in catecholamine release acting through the Y 1 receptor. This work suggests that NPY is involved in the regulatory loop between the immune and the adrenal system in some pathophysiological conditions where plasmatic IL-1β increases, like in sepsis, rheumatoid arthritis, stress or hypertension.

Myristoylated Alanine-rich C Kinase Substrate-mediated Neurotensin Release via Protein Kinase C-δ Downstream of the Rho/ROK Pathway

Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-alpha and -delta, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-delta siRNA, ROKalpha siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-delta and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-delta was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-delta downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-delta proteins, in stimulated gut peptide secretion.

Differential effects of FK506 and cyclosporin A on catecholamine release from bovine adrenal chromaffin cells

1 The effects of the immunosuppressants, tacrolimus (FK506) and cyclosporin A (CsA), on catecholamine (CA) release were examined in cultured bovine adrenal chromaffin cells. 2 In intact cells, FK506 (1-30 microM) inhibited CA release stimulated by acetylcholine (ACh; 100 microM), 1,1-dimethyl-4-phenyl-piperazinium (DMPP, 10 microM) or high K+ (40 mM). CsA (1-30 microM) had a little inhibitory effect on the ACh- or DMPP-stimulated CA release, whereas it enhanced the high K(+)-stimulated CA release. 3 In beta-escin-permeabilized cells, FK506 inhibited CA release stimulated by Ca2+ (1 and 10 microM) in the presence and absence of MgATP (2 mM). CsA induced CA release under Ca(2+)-free condition and enhanced the Ca(2+)-stimulated CA release in the presence and absence of MgATP. 4 It is known that the Ca(2+)-dependent exocytosis involves at least two distinct steps, ATP-requiring priming stage and ATP-independent fusion step in adrenal chromaffin cells. Therefore, it is suggested that FK506 inhibits the Ca(2+)-dependent exocytosis probably at the fusion step whereas CsA induces CA release from bovine adrenal chromaffin cells.