Accumulation Of Catecholamines Research Articles

The quantal secretion of catecholamines is impaired by the accumulation of β‐adrenoceptor antagonists into chromaffin cell vesicles

The delayed onset of certain effects of antagonists of beta-adrenoceptors (beta-blockers), such as lowering arterial blood pressure (several days), cannot be explained solely by their effects on beta-adrenoceptors, an action that occurs within minutes. Although several mechanisms have been proposed, none of them explain this temporal delay. This work aimed at providing a new explanation based on the interference of these drugs with the functional accumulation of catecholamines within neurosecretory vesicles. We used the simultaneous on-line monitoring of catecholamine and labetalol release from bovine isolated chromaffin cells and from rat perfused adrenal glands, as well as single cell amperometry, intracellular electrochemistry, patch amperometry and HPLC. Using amperometry, three beta-blockers, labetalol, atenolol and propranolol, reduced the quantal size of secretory events in chromaffin cells, accompanied by a slowing down of exocytosis. By patch amperometry, we found that treatment with beta-blockers also increases the chromaffin vesicle volume, thereby creating a functional dilution of catecholamines. Experiments with intracellular electrochemistry show that vesicles cannot uptake new catecholamines. There was progressive accumulation of labetalol in secretory vesicles of bovine adrenal chromaffin cells, and this beta-blocker was co-released with catecholamines from rat and bovine chromaffin tissues. We propose that beta-blockers are progressively concentrated into sympathetic secretory vesicles, and interfere with the storage of catecholamines and are co-released with the natural transmitters, resulting in a decrease in the sympathetic tone. This could explain the delayed onset of the hypotensive effects of beta-blockers.

Chromogranin B Gene Ablation Reduces the Catecholamine Cargo and Decelerates Exocytosis in Chromaffin Secretory Vesicles

Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb(+/+) [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (approximately 30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with L-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that L-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.

Contribution of catechol O-methyltransferase to the removal of accumulated interstitial catecholamines evoked by myocardial ischemia

Catechol O-methyltransferase (COMT) plays an important role for clearance of high catecholamine levels. Although myocardial ischemia evokes similar excessive catecholamine accumulation, it is uncertain whether COMT activity is involved in the removal of accumulated catecholamines evoked by myocardial ischemia. We examined how COMT activity affects myocardial catecholamine levels during myocardial ischemia and reperfusion. We implanted a dialysis probe into the left ventricular myocardial free wall and measured dialysate catecholamines levels in anesthetized rabbits. Dialysate catecholamine levels served as an index of myocardial interstitial catecholamine levels. We introduced myocardial ischemia by 60 min occlusion of the main coronary artery. The ischemia-induced dialysate catecholamines levels were compared with and without the pretreatment with entacapone (COMT inhibitor, 10 mg/kg, i.p.). Acute myocardial ischemia progressively increased dialysate catecholamine levels. Acute myocardial ischemia increased dialysate norepinephrine (NE) levels (20,453 ± 7186 pg/ml), epinephrine (EPI) levels (1724 ± 706 pg/ml), and dopamine (DA) levels (1807 ± 800 pg/ml) at the last 15 min of coronary occlusion. Inhibition of COMT activity by entacapone augmented the ischemia-induced NE levels (54,306 ± 6618 pg/ml), EPI levels (2681 ± 567 pg/ml), and DA (3551 ± 710 pg/ml) levels at the last 15 min of coronary occlusion. Myocardial ischemia evoked NE, EPI, and DA accumulation in the myocardial interstitial space. The inhibition of COMT activity augmented these increments in NE, EPI, and DA. These data suggest that cardiac COMT activity influences on the removal of accumulated catecholamine during myocardial ischemia.

Intracellular Patch Electrochemistry: Regulation of Cytosolic Catecholamines in Chromaffin Cells

Alterations in the cytosolic pool directly affect neurotransmitter synthesis and release and are suggested to be key factors in various neurodegenerative disorders. Although this cytosolic pool is the most metabolically active, it is miniscule compared with the amount of vesicular transmitter and has never been quantified separately. Here, we introduce intracellular patch electrochemistry (IPE), a technique that for the first time provides direct measurements of cytosolic oxidizable molecules in single mammalian cells. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it preferentially measures catecholamines. In cultured chromaffin cells, the total cytosolic catechol concentration was 50-500 microm, of which approximately 10% were catecholamines. Reserpine, a vesicular monoamine transporter inhibitor, had no effect on the catecholamine pool but increased total catechols by fourfold to fivefold. Combined with pargyline, a monoamine oxidase inhibitor, reserpine increased catecholamine levels in the cytosol by approximately sixfold. Amphetamine induced a transient approximately fivefold accumulation of cytosolic catecholamines and a slow increase of total catechols. In cells incubated with 3,4-dihydroxy-L-phenylalanine (L-DOPA), catecholamines increased by approximately 2.5-fold and total catechols increased by approximately fourfold. Cytosolic catecholamines returned to control levels <or=10 min after L-DOPA withdrawal, whereas total catechols remained approximately twofold elevated even after a 1.5 hr incubation in L-DOPA-free media. Our data indicate that cytosolic catecholamines are strictly maintained at a defined level, and drug-induced increases in their concentrations lead to the accumulation of other catecholamine derivatives, such as DOPAC and 3,4-dihydroxyphenylethyleneglycol. These derivatives reside in the cytosol for hours after treatment and may be an underlying cause of drug-related cytotoxicity.

Smaller muscle ATP reduction in women than in men by repeated bouts of sprint exercise.

It was hypothesized that the reduction of high-energy phosphates in muscle after repeated sprints is smaller in women than in men. Fifteen healthy and physically active women and men with an average age of 25 yr (range of 19-42 yr) performed three 30-s cycle sprints (Wingate test) with 20 min of rest between sprints. Repeated blood and muscle samples were obtained. Freeze-dried pooled muscle fibers of types I and II were analyzed for high-energy phosphates and their breakdown products and for glycogen. Accumulation of plasma ATP breakdown products, plasma catecholamines, and blood lactate, as well as glycogen reduction in type I fibers, was all lower in women than in men during sprint exercise. Repeated sprints induced smaller reduction of ATP and smaller accumulation of IMP and inosine in women than in men in type II muscle fibers, with no gender differences in changes of ATP and its breakdown products during the bouts of exercise themselves. This indicates that the smaller ATP reduction in women than in men during repeated sprints was created during recovery periods between the sprint exercises and that women possess a faster recovery of ATP via reamination of IMP during these recovery periods.

Regional distribution and control of tyrosine hydroxylase activity in the quail brain

Tyrosine hydroxylase (TH) activity, the rate-limiting step in the synthesis of catecholamines, was quantified in the preoptic area-hypothalamus of adult male Japanese quail by a new assay measuring the tritiated water production from 3,5-[ 3H]-L-tyrosine. Maximal levels of activity were observed at a 20–25 μM concentration of substrate, with more than 50% inhibition of the activity being recorded at a 100 μM concentration. TH activity was linear as a function of the incubation time during the first 20 min and maximal at a pH of 6.0. TH was heterogeneously distributed in the quail brain with highest levels of activity being found (in decreasing order) in the mesencephalon, diencephalon, and telencephalon. Given the large size of the telencephalon, this is the brain area that contains, as a whole, the highest level of enzyme activity. TH inhibitors that have been well-characterized in mammals, such as 3-iodo- l-tyrosine and l-α-methyl-p-tyrosine (AMPT) completely inhibited the enzyme activity at a 100 μM concentration. In mammals, the accumulation of catecholamines exerts a negative feedback control on TH activity. Similar controls were observed in the quail brain. Two inhibitors of the DOPA decarboxylase that should lead to accumulation of DOPA depressed TH activity by 60% or more, and the inhibitor of the dopamine β-hydroxylase, fusaric acid that should cause an accumulation of dopamine, suppressed 90% of the TH activity. The addition of exogenous DOPA, dopamine, or norepinephrine to the brain homogenates also strongly inhibited TH activity, independently confirming the feedback effects of the enzyme products on the enzyme activity. These data demonstrate that TH activity in the quail brain is heterogeneously distributed and acutely regulated, as it is in mammals, by the accumulation of its products and of the derived catecholamines.

Cellular and pathophysiological mechanisms of ventricular arrhythmias in acute ischemia and infarction.

Ventricular arrhythmias in the setting of acute myocardial ischemia and infarction remain a serious health problem because of their sudden and unpredictable nature and their potentially grave results. Electrophysiological changes that may be responsible for these arrhythmias have been described in cardiac cells and in ischemic tissue. Experimental models have played a major role in elucidating the diversity of potential mechanisms for these arrhythmias. Increases in extracellular K+, the presence of toxic metabolites, and the accumulation of catecholamines in ischemic tissue all appear to have a role in arrhythmogenesis. The autonomic nervous system also appears to play a major role in these arrhythmias. With increased understanding of the pathophysiology underlying these arrhythmias, prevention can be enhanced and therapy can be better targeted.

Ephedrine-Induced Coronary Artery Vasospasm in a Patient with Prior Cocaine Use

Ephedrine-Induced Coronary Artery Vasospasm in a Patient with Prior Cocaine Use

Reaction of adrenal medulla to extreme factors of various nature

Experiments on mice show that primary decrease in the catecholamine content in the adrenals 6 h and 1–2, and 4 days after immobilization stress is followed by catecholamine accumulation in the adrenal medulla. Practically the same dynamics within days 1–4 is observed after administration of a high dose of 5-fluorouracil; however, the restoration of the initial catecholamine level in mice treated with the cytostatic takes more than in stressed animals. These data suggests that high doses of 5-fluorouracil induce nonspecific activation of the sympathoadrenal system, presumably leading to adaptation to cytotoxic effects of 5-fluorouracil.

Enhancement by F-actin of MgATP-dependent dopamine uptake into isolated chromaffin granules.

Interaction of chromaffin granules with the microfilament network in the cytoplasmic space of adrenal medullary cells has been proposed, but the influence of chromaffin granule-microfilament interaction on granule function has not yet been studied. In the present study, the effect of F-actin on dopamine uptake into isolated chromaffin granules was examined. The MgATP-dependent component of dopamine uptake was significantly enhanced by F-actin. In contrast, catecholamine efflux from the granules was not significantly altered by F-actin under the conditions in which the enhancement of MgATP-dependent dopamine uptake was observed. These findings suggest that the interaction of chromaffin granules with the microfilament network results in the enhancement of MgATP-dependent dopamine uptake into the granules, thus modulating the accumulation and storage of catecholamines in the adrenal medullary cell.

A dopamine transporter in human erythrocytes: modulation by insulin.

Red blood cells are capable of transport and accumulation of catecholamines. The aim of this paper is to characterize the catecholamine transport system in the human red blood cell and in particular that of dopamine. Dopamine, noradrenaline and adrenaline enter the red blood cell by a similar process, which shows saturation kinetics with Vmax values of 0.54 +/- 0.12, 0.48 +/- 0.08 and 0.63 +/- 0.13 mumol (1 cells)-1 min-1, respectively, and K(m) values of 15.62 +/- 1.19, 5.81 +/- 1.19 and 12.00 +/- 2.97 nM, respectively. Observations based on the dependence of dopamine influx on the transmembrane H+ gradient, and the effect of transport inhibitors such as DMA (dimethyl-amiloride), DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulphonic acid), reserpine, GBR 12909 (1-(2-(di(4-fluoro-phenyl)-methoxy)-ethyl)-4-(3-phenylpropyl)piperazine) , GBR 12935 (1-(2-(diphenyl-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine), and cyanine suggest that catecholamine transport is not mediated by the Na(+)-H+ exchanger, the anion exchanger or a system similar to that responsible for dopamine uptake in either synaptosomes or the proximal tubule. However, choline inhibits the influx of dopamine with an IC50 value of 17 microM and stimulates the efflux of dopamine with a K(m) value of 8.20 microM. These results strongly suggest that dopamine is transported by the choline exchanger previously reported to be present in red blood cells. Probenecid inhibits dopamine uptake with an IC50 of 0.63 microM. The presence of insulin receptors in human red blood cells, and the relationship between insulin and catecholamine levels in the plasma led us to investigate the effect of insulin on catecholamine transport. In fasting subjects, dopamine, adrenaline and noradrenaline influxes were higher than in fed subjects. Furthermore, the addition of exogenous insulin to red blood cells from fasting subjects significantly reduced the influx of catecholamines while no effect was observed when insulin was added to red blood cells obtained from fed subjects. The present study shows that catecholamines, and in particular dopamine, are transported in red blood cells via an exchanger which is possibly the choline transport system. The activity of this transporter is regulated by insulin. These results support a role for red blood cells as a storage pool for circulating catecholamines.

Cocaine: a review of its toxic actions on cardiac function.

It has become increasingly apparent that cocaine abuse can provoke lethal cardiac events, including myocardial infarction and ventricular fibrillation. The mechanisms responsible for these cardiotoxic actions of cocaine largely remain to be determined. Cocaine has two primary pharmacological properties that can adversely affect the heart and vasculature. Cocaine acts both as a local anesthetic (sodium and potassium channel blockade) and as a powerful cardiac stimulant that accentuates the actions of the sympathetic nervous system (inhibition of central and peripheral neuronal catecholamine uptake). The local anesthetic properties could impair impulse conduction, as well as elicit inhomogeneities in repolarization (refractory period), which creates an ideal substrate for reentrant arrhythmias. In addition, high doses of cocaine can depress contractile function due to inhibition of sodium/calcium exchange that results from decreased sodium influx (local anesthetic action). These actions are particularly obvious when sympathomimetic effects of cocaine are blunted. In a similar manner, the cocaine-induced accumulation of catecholamines potentiates the activation of alpha- and beta-adrenergic receptors, which can provoke coronary vasospasm (myocardial ischemia and infarction), increased contractile force (increased metabolic demand), and cardiac arrhythmias. The activation of adrenergic receptors will elicit a cascade of second messengers, ultimately provoking an increase in cytosolic calcium. These elevations in cytosolic calcium can provoke oscillations in membrane potential, triggering sustained action potential generation and extrasystoles. In particular, activation of the alpha IA-adrenergic receptor subtype and corresponding increase in calcium influx via voltage sensitive (L type) channels may play a critical role in the genesis of malignant arrhythmias. Thus, the adrenergic and local anesthetic properties of cocaine could act synergistically to elicit toxic actions on the heart.

Plasma Erythrocyte Relationship of Catecholamines in Human Blood

The equilibrating process of catecholamines (CAs) between plasma and red blood cells (RBC) was studied by measuring their erythrocyte/plasma concentration gradient (E/P ratio); ratio of E/P > 1 for a given amine was considered the consequence of its accumulation in or on RBC. We studied in vitro human blood obtained from 9 polycythemic patients from whom blood was drawn in control condition and in response to loading with exogenous CAs. Preliminary study showed a lack of difference in results obtained in these patients from those in 9 healthy volunteers and confirmed that RBC accumulate dopamine (DA) and epinephrine (EPI) but not norepinephrine (NE). When a moderate amount of exogenous CAs was added, plasma and RBC concentrations were increased, with a delay between the two compartments indicated by a decrease in the E/P ratio of DA and EPI. When a large amount of exogenous CAs was added, their RBC concentrations were in direct proportion to their plasma concentrations. Thus, the kinetics of CA equilibration between plasma and RBC appears to be dependent on their chemical structure (DA is more easily accumulated than NE) and their plasma concentrations. Physiologically, the E/P ratio of DA was significantly greater than 1, suggesting that RBC maintained their capacity to accumulate DA even when DA plasma concentration was very high. Additional studies demonstrated that accumulation of CAs in or on RBC is a reversible process, inhibited by cold temperature and increased when blood pO2 is drastically reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory Effect of IL-1β on Catecholamine Secretion from Cultured Bovine Adrenal Medullary Cells

We investigated the effect of recombinant human interleukin-lβ (IL-lβ) on catecholamine secretion from cultured bovine adrenal medullary cells. Treatment of cultured cells with IL-1β (10 ng/ml) for 24 hr caused an increase in accumulation of catecholamines in the cultured medium. The accumulation of catecholamines stimulated by IL-1β was observed in time (4 - 48 hr)- and concentration (3 - 30 ng/ml)-dependent manners. The stimulatory effect of IL-1β (10 ng/ml) was completely inhibited by recombinant human IL-1 receptor antagonist (1 μg/ml). IL-1β had little effect on [ 3H]norepinephrine uptake to cultured cells. These results suggest that IL-1β stimulates catecholamine secretion through activation of IL-1 receptors in adrenal medullary cells.

Pertussis toxin inhibits noradrenaline accumulation by bovine adrenal medullary chromaffin cells.

Bovine adrenal medullary chromaffin cells maintained in tissue culture accumulated [3H]-noradrenaline by a high affinity, Na(+)-dependent, desipramine-sensitive process. The accumulation was linear with time (1-90 min) and had an apparent Km of 0.52 +/- 0.24 mumol/l and Vmax of 1.70 +/- 0.48 pmol/(10(5) cells.15 min). Pretreatment of the cells with the ADP-ribosylating agent pertussis toxin resulted in a reduction in the Vmax [0.81 +/- 0.39 pmol/(10(5)cells.15 min)] but no significant change in the apparent affinity (Km = 0.42 +/- 0.07 mumol/l). This inhibition of [3H]noradrenaline accumulation was distinct from that produced by the vesicular transport inhibitor reserpine. Pertussis toxin inhibition probably did not arise through an indirect action on the Na(+)-gradient because while, as expected, Na+,K(+)-ATPase inhibition reduced [3H]noradrenaline accumulation, pertussis toxin pretreatment always caused a further significant reduction even in the presence of maximally effective concentrations of ouabain. Stimulation of the cAMP-protein kinase A system by forskolin or 8-bromocyclic AMP also caused a reduction in [3H] noradrenaline accumulation but again pertussis toxin pretreatment always resulted in a further reduction. Thus, the data provide evidence for a pertussis toxin-sensitive element in the catecholamine accumulation process and are consistent with an action at a site directly associated with the transporter itself rather than with an indirect action via secondary processes.

Ventricular arrhythmias, sudden death, and silent myocardial ischemia

T HE ASSOCIATION between acute ischemia and ventricular arrhythmia in experimental animals is well established. Kaplinsky et al’ found two periods of early ventricular arrhythmia within 30 minutes of coronary occlusion in dogs. Janse et al* described two mechanisms of ventricular arrhythmia during acute experimental ischemia. At the normal side of the ischemic border they found a focal mechanism, induced possibly by injury currents in the normal Purkinje fibers, whereas in the ischemic myocardium they found macroand microreentry ventricular arrhythmia. Coronary occlusion in cats3 caused increased conduction time through the myocardium and epicardial regions, asynchronous depolarization and shortening of the refractory period, accompanied by ventricular arrhythmias. Kabell et al4 demonstrated that ischemia induced by reduction of collateral flow to an infarcted area in experimental animals, induced fractionation and delay of the electrogram in the infarcted zone and a higher frequency of pacing-induced ventricular tachycardia (VT). The effect of repeated 5-minute periods of ischemia followed by reperfusion on ventricular arrhythmia was assessed by Shiki and Hearse5 in rats; during reperfusion all animals exhibited VT and 83% of them exhibited ventricular fibrillation (VF). After conversion to sinus rhythm and following recovery, they were again subjected to 5-minute periods of ischemia and reperfusion. Following a short recovery period from the initial bout of ischemia only 8% to 17% of the experimental animals developed VT and none had VF, whereas longer recovery periods were associated with a progressive return of vulnerability. Thus, a very short period of ischemia and reperfusion “preconditioned” and protected the myocardium from reperfusion arrhythmias, but for only a short period of time. Recent evidence suggests that platelet activation may contribute to arrhythmogenesis during myocardial ischemia. Flores et al6 found that in ischemic myocardium platelets increased conduction time and reduced action potential duration. Further support for the role of platelets in inducing life-threatening arrhythmias was provided by Davies et al7 who described platelet aggregation as a frequent finding in patients with unstable angina suffering sudden ischemic cardiac death. The association between catecholamines, ischemia, and arrhythmia is a complex one. Within 10 minutes of ischemia the noradrenaline concentration in the myocardium is increased.8l” The accumulation of catecholamines in the extracellular space during ischemia in viable myocardium can cause malignant arrhythmia and acceleration of cell damage.‘O High doses of epinephrine decrease the resting membrane potential and action potential amplitude, slow conduction, and produce undirectional block; all these alterations predispose to reentrant arrhythmias. Epinephrine can also induce triggered activity, the underlying cause of automatic arrhythmias. i1,12 The importance of the sympathetic system during ischemia-induced arrhythmias is further substantiated by the protective effects of beta blockers both against myocardial ischemia and necrosisi3s14 and cardiac arrhythmias. 15.1h In human hearts no enhanced cardiac release of noradrenaline during coronary angioplastyinduced ischemia could be detected by sampling of coronary sinus blood.17 An association between ischemia and arrhythmias in humans was demonstrated by Garan et al’s in 17 patients with multivessel disease who survived out-ofhospital cardiac arrest. Before bypass surgery all developed VT during pacing-induced ischemia; after surgery VT or VF could not be induced in 10 patients, suggesting that ischemia was a major contributing factor for arrhythmia in these patients. Morady et al’” performed electrophysiological stimulation in 15 survivors

Plasma catecholamines and accumulation of adrenaline in the atrial cardiac tissue of aquacultured Atlantic salmon (Salmo salar) during stress

The effects of stress on plasma catecholamines (CA) and capacity for tissue accumulation of CA were studied in cardiac and skeletal muscle of cultured Atlantic salmon (Salmo salar L.). Adrenaline (A) and noradrenaline (NA) were quantified by high performance liquid chromatography.Plasma A and NA levels were 56±10 nmoll−1 and 77±17 nmoll−1 (±s.e.m.), respectively, in a control group living under normal rearing conditions in a fish farm. Following a ±3 h period of pre‐slaughter crowding and handling in the fish farm, plasma A reached 221 ± 72 nmol1−1 with no increase in plasma NA. An 0.5 h period of struggling out of water led to even higher level of plasma A (480 ± 89 nmol1−1), without change in NA.Skeletal muscle was low in CA (A, 0.07 ± 0.02 and NA, 0.06 ± 0.01 nmol g−1 wet wt). Tissue CA was higher in the atrium (A, 0.47 ± 0.04 and NA 0.94 ± 0.10 nmol g−1) than in the ventricle (A, 0.25 ± 0.03 and NA, 0.30 ± 0.02 nmol g−1). The 0.5 h period out of water and the 0.5‐3 h period of pre‐slaughter crowding led to accumulation of A, but not NA, in the atrium.These data show that A, released during stress, accumulates in an undegraded form in the atrial tissue of the Atlantic salmon. This suggests a potent uptake mechanism for A in the atria presumably in the sympathetic nerve terminals. The acumulation of A in the atrium appears to reflect the period of high plasma A during stress.

The avian allantois: A depot for stress-released catecholamines

Plasma and amniotic and allantoic fluid of 10- and 14-day-old chicken embryos contain free dopamine (DA), norepinephrine (NE), and epinephrine (E). Compared with postnatal chickens, concentrations of DA and E in the plasma are very high, and they are even higher in the allantoic fluid. In contrast, the allantoic concentration of NE is below the plasma level. In the amniotic fluid, the concentrations of all three catecholamines (CAs) are below the plasma levels. High concentrations of DA and E in the allantoic fluid after opening of the egg shell decline during the following 24 hr, which indicates that they are due to stress. Asphyxia, handling, disturbance of allantoic fluid, and cooling are also perceived as stress and are followed by immediate accumulation of CAs in the allantoic fluid. DA and E respond to stress in like manner, while NE often responds with an opposite trend. It appears that the avian allantois, in addition to its role in respiration and urea disposal, also serves the instant CA removal from the circulation. Both the amniotic and the allantoic membranes of the chicken should be ideal models for the study of CA transport mechanisms.

Characterization of the norepinephrine uptake system and the role of norepinephrine in the expression of the adrenergic phenotype by quail neural crest cells in clonal culture

This study investigates the role norepinephrine (NE) may play in regulating the differentiation of quail neural crest cells into sympathoadrenal cells. Cues originating from the embryonic microenvironment are thought to play an important role during development. It is conceivable that NE has a positive regulatory function because adrenergic expression by quail neural crest cells in clonal culture can be inhibited by NE uptake inhibitors such as desipramine (DMI). This possibility is further supported by the notion that in the avian embryo presumptive adrenergic neural crest cells are likely to encounter catecholamines shortly after they have acquired the NE uptake mechanism. Our present data indicate that neural crest cells in clonal culture express a high affinity NE uptake system that can be inhibited by desipramine. As in the embryo, it appears before noticeable levels of catecholamines are accumulated by neural crest cells, as judged by formaldehyde-induced catecholamine fluorescence (FIF). A comparison of the time course of appearance of different adrenergic markers suggests that immunoreactivity against the biosynthetic enzyme tyrosine hydroxylase (TH) may appear first, and that it is followed very closely by the appearance of detectable levels of dopamine-β-hydroxylase (DBH) and the NE uptake mechanism. Accumulation of catecholamines (FIF) is observed last. Addition of exogenous NE leads to an increase in adrenergic expression in vitro as judged by an increase in the number of colonies containing FIF-positive cells as well as cells expressing the biosynthetic enzymes TH and DBH. This suggests that exogenous NE can play a positive regulatory role in the differentiation of quail neural crest cells intossympathoadrenal cells.

Ontogenesis of the hypothalamic catecholaminergic system in rats: Synthesis, uptake and release of catecholamines

The development of the hypothalamic catecholaminergic system during ontogenesis in rats has been studied with glyoxylic acid histofluorescent method in vivo and with the isotopic biochemical technique in vitro. It has been demonstrated that at the 15th fetal day the catecholaminergic system was functionally inactive at least in its ability for the uptake and K +-stimulated release of catecholamines. Since the 16th fetal day, hypothalamic neuronal elements gained an ability for synthesis of catecholamines, their specific uptake and K +-evoked release. Over the subsequent two days, the intensity of the fluorescent intraneuronal product rose considerably showing the increase of either synthesis or accumulation of catecholamines. Simultaneously, the value of the uptake and K +-stimulated release of the exogenous radioactively-labelled dopamine increased significantly. The intensity of the fluorescence of the hypothalamic neuronal elements dropped from 20th fetal until the ninth postnatal day, whereas the specific uptake doubled over the same period reaching itsaadult level. By the 21st postnatal day the reaccumulation of the fluorescent product occurred.