Insulin-like growth factor-I affects amino compounds in the fluids of the chicken embryo.

The concentration differences of more than 40 amino acids and related compounds in the amniotic fluid, allantoic fluid, and plasma of the chicken embryo are maintained by specific barriers. Since the amniotic and allantoic membranes are not innervated, we proposed that these barriers are controlled by hormones. Specific effects of insulin and prolactin on the amino compounds in the three fluids confirmed this hypothesis and raised the question of the possible role of growth factors. Application of insulin-like growth factor-I (IGF-I) to the chorioallantoic membrane of day 13 chicken embryos caused the following concentration changes in 41 amino compounds measured 1 and 2 h later: (1) in the amniotic fluid, an increase of 40 compounds, regardless of the presence or absence of a concomitant stress effect on these compounds; only NH3 was not affected; (2) in the allantoic fluid, a decrease of reduced glutathione (GSH) and anserine, and an increase of NH3; (3) in the plasma, a decrease of 24 compounds. Within the same time frame, stress caused in the amniotic fluid a drop of the concentration of 29, and an increase of 5, amino compounds; IGF-I reversed the stress effect on all 29 compounds the concentrations of which had dropped and enhanced the stress-induced increase of the other 5 compounds. In the allantoic fluid, stress induced an increase of GSH; IGF-I reversed this effect. In the plasma, stress caused an increase of 9 compounds; IGF-I counteracted the increase in 7 cases. These findings indicate new and unexpected roles of IGF-I in the prenatal regulation of amino compounds.

Hormonal effects on amino acids and related compounds in plasma, amniotic fluid, and allantoic fluid of the chicken embryo.

So far, more than 40 free amino acids and related compounds have been identified in plasma, amniotic fluid, and/or allantoic fluid of the 13-day chicken embryo. Concentration differences, and greatly varying behavior of these compounds under experimental conditions, revealed the presence of specific barriers among the three fluids. We tested the hypotheses that (1) the absence of an innervation of amnion and allantois indicates a hormonal control of their barriers, and (2) changes in the concentrations of certain amino compounds in the three fluids indicate anabolic or catabolic actions of hormones. Insulin, prolactin, and stress caused complex changes of the concentrations of amino compounds in all three fluids within 30 min. Some of these changes indicated breakdown of embryonic tissues, while others must have been due to transfer of amino compounds among the three fluid compartments. However, there was no significant effect on the glucose concentration in any of the three compartments under any of the experimental conditions. This is the first demonstration of hormonal effects on the amino compounds in the extraembryonic fluids of nonmammalian amniotes.

Impact of exogenous amino acids on endogenous amino compounds in the fluid compartments of the chicken embryo.

Plasma, allantoic and amniotic fluid of the 13-day chicken embryo contain numerous free amino acids and related compounds. Of these, 40 were investigated using an HPLC-fluorometric technique. The concentration differences of the amino compounds between the fluid compartments are maintained by three bidirectional barriers, i.e. a blood/allantoic barrier, a blood/amnion barrier and an allantois/amnion barrier. Intraallantoic injection of 4.5 mumol/egg of asparagine (ASN), aspartic acid (ASP), valine (VAL) or serine (SER) revealed a strong allantois/blood barrier for these compounds. In contrast, there was equilibration between allantoic and amniotic fluid for ASN, ASP and SER, and an upward trend of the VAL concentration in the amniotic fluid, due to an 'overspill' from the allantois. The injections also affected endogenous amino compounds in all three fluid compartments. Asparagine had the most varied effects, including a strong drop of ten plasma amino acids. After all four types of injection, a number of endogenous amino compounds equilibrated between allantois and the normally hyporegulated amnion. Since allantois and amnion are non-innervated, the selective changes of the barriers and the drop of plasma amino acids must be mediated by so far unidentified humoral messengers.

Electrostimulation of catecholamine release in the eel: modulation by antagonists and autocrine agonists.

The innervated chromaffin cells of the eel (Anguilla rostrata) release norepinephrine (NE) and epinephrine (E), while a component of the macrovascular wall releases dopamine (DA). The release of the three catecholamines is governed by complex controls which include adrenergic, nicotinergic, muscarinergic, and opioid mechanisms. To gain insight into the interactions between neural and autocrine factors in stimulated catecholamine release, we investigated the effect of adrenergic (phentolamine and propranolol) and muscarinergic (atropine) receptor antagonists, and of autocrine opioids (met-enkephalin, codeine, and morphine) on electrostimulated catecholamine secretion in situ. The hind brain (close to the root of nerve IX) of anesthetized eels was stimulated at four different time points, and segments of the posterior cardinal vein or the caudal vein were perfused with a saline solution, with or without test substances. Electrostimulation (30 s) four times within a total study duration of 14 min increased the release of DA, NE, and E into the perfusate of the cardinal vein. The vessel contains the innervated adrenomedullary equivalent. In the noninnervated caudal vein electrical stimulation had no impact on total DA release, while there was a slight decrease of NE release and a slight increase of E release. In the cardinal vein, both the alpha-adrenergic receptor antagonist phentolamine and the beta-adrenergic receptor antagonist propranolol strongly reduced the effect of electrostimulation on catecholamine release. Met-enkephalin reduced the release of all three catecholamines to a similar degree; its impact on NE release was especially strong. Codeine reduced the catecholamine release moderately, while morphine had no effect. Atropine reduced the release of all three catecholamines in a pattern similar to that of met-enkephalin. The findings on the posterior cardinal vein indicate that neurally stimulated NE and E release (1) involves autocrine/paracrine adrenergic mechanisms, (2) involves a muscarinergic mechanism, and possibly also endogenous codeine and morphine; and (3) is antagonized by met-enkephalin. The findings on the caudal vein are further evidence that macrovascular DA release is not under direct neural control.

Cholinergic control of catecholamine release in the eel.

The perifused posterior cardinal vein of the American eel (Anguilla rostrata) releases spontaneously dopamine (DA), norepinephrine (NE), and epinephrine (E). NE and E are secreted by innervated chromaffin cells, while DA is most likely released from a component of the vascular wall. Stimulation with acetylcholine strongly enhances the release of DA and E, and to a lesser degree the release of NE. Nicotine stimulates the release of all three catecholamines. Muscarine reduces the basal release of NE. Muscarine does not prevent nicotinic stimulation of NE and E release, but abolishes the nicotine effect on DA release. The muscarinic antagonist atropine stimulates the release of NE, but not of DA and E. The beta-adrenergic receptor antagonist propranolol suppresses the acetylcholine-stimulated release of NE and E, and reduces the DA response. From these findings, it appears that (1) nicotinic receptors regulate NE and E secretion from the chromaffin cells, (2) muscarinic receptors inhibit basal NE release, and (3) acetylcholine-stimulated release of NE and E requires the interaction with adrenergic receptors. On the other hand, DA release involves both nicotinic and adrenergic receptors, while the reduction of nicotine-stimulated (but not basal) DA release involves muscarinic receptors.

Opioid effects on macrovascular dopamine release.

Both alkaloid opiates and met-enkephalin occur in vertebrate chromaffin cells, where they affect catecholamine (CA) secretion. Since the large blood vessels of the eel and the rat release dopamine (DA) from as yet unidentified source(s), we studied the impact of alkaloid opiates and met-enkephalin on the secretion of DA from three macrovessels of the American eel (Anguilla rostrata) in a perifusion system. Codeine, morphine, and met-enkephalin increased the release of DA from both the ventral aorta and the caudal vein. The antagonist naloxone stimulated DA release from the caudal vein, but had no impact on release from the ventral aorta. Only codeine had a significant effect on DA release from the posterior cardinal vein. These findings show that the DA release from the macrovessels is sensitive to opioid substances, and they suggest that the antagonistic effects between alkaloid opiate and opioid peptide, seen in other systems, are absent in large blood vessels. Furthermore, the "unorthodox" stimulatory effect of naloxone in the caudal vein raises the question of as yet unidentified receptor and/or effector systems.

Impact of ethanol stress on components of the allantoic fluid of the chicken embryo.

Recent studies showed that the allantoic fluid of the chicken embryo is a depot for stress-released catecholamines and many free amino acids and related compounds, and that it is separated from plasma and the amniotic fluid by selective barriers. To gain further insights into the functions of the allantois and its barriers, we studied the impact of stress (intra-allantoic injection of 0.1 ml ethanol) on 39 free amino acids and related compounds of the allantoic fluid. Using an HPLC-fluorometric method, we found that the concentration of seven substances was significantly increased 20 min after injection of ethanol, and back to control levels within 40 minutes. Five of these compounds (asparagine, alanine, leucine, tyrosine, lysine) had previously been shown to occur in plasma at concentrations above those in the allantoic fluid. However, taurine and phosphoethanolamine increased in the allantoic fluid even though their concentrations in plasma tended to be lower than in allantoic fluid. These findings (1) reveal the existence of complex embryonic/extraembryonic autoregulations, and (2) raise the question of the regulatory mechanisms involved in the transfer of substances across the allantoic barrier(s).

Regulation of substances in allantoic and amniotic fluid of the chicken embryo.

Traditionally, the avian allantois has been considered a respiratory organ and a dumping ground for metabolic wastes. We tested the hypothesis that the allantoic fluid is also a depot for free amino acids and related compounds. To gain further insight in the specific role of the allantoic fluid, we included plasma and the amniotic fluid in this study. The work was carried out in 13- and 14-day-old chicken embryos. Using an HPLC-fluorometric technique, 40 of the 41 amino acids and related compounds investigated were detected. The amniotic fluid contained 32 compounds, while plasma and allantoic fluid contained 38 and 39 compounds, respectively. The glucose concentration was determined with a hexokinase technique. It was highest in plasma and lowest in the amniotic fluid. We identified three barriers that hyper- and hyporegulate a number of compounds: (1) a blood/allantois barrier, (2) a blood/amnion barrier, and (3) an allantois/amnion barrier. Compared with plasma and allantoic fluid, the amniotic fluid is a mostly hyporegulated environment.

Macrovascular dopamine release.

In an animal model, the American eel, perifused elastic arteries and large veins, but not the heart and organs with extensive microvascular supply (gills and opisthonephric kidney), release spontaneously free dopamine. Only the region of the cardinal vein, which contains the adrenomedullary equivalent, also releases norepinephrine (NE) and epinephrine (E). Ca2+, KCl, and E stimulate dopamine release from the ventral aorta and caudal vein, indicating that this phenomenon is due to secretion and not to washout. E also stimulates NE release from the ventral aorta and caudal vein. In the rat, both aorta and vena cava spontaneously release dopamine and NE. Thus dopamine secretion from large blood vessels may be general in vertebrates. The dopamine response to high physiological concentrations of E in vivo and in vitro suggests that macrovascular dopamine may be involved in local stress responses.

Release of amino acids and related compounds from the adrenal equivalent and caudal vein of the eel in vitro.

The release of catecholamines and cortisol from the perifused adrenal region and caudal vein of the eel (Anguilla rostrata) was compared with the release of 39 amino acids and related compounds. Dopamine, norepinephrine and epinephrine were present in all perifusates of the adrenal region. Dopamine release from the caudal vein exceeded that from the adrenal region, and norepinephrine and epinephrine were not detected. Cortisol was present in the perifusate of the adrenal region but virtually absent in caudal vein perifusate. Of the six substances with known or suspected neurotransmitter function, taurine, aspartate, glutamate, glycine and alanine were present in all or almost all samples from both the adrenal equivalent and the caudal vein. gamma-aminobutyric acid (GABA) was detected in a few samples from either preparation. The release of taurine and phosphoethanolamine may be linked to that of norepinephrine and epinephrine. Adrenocorticotropic hormone (ACTH) enhanced the release of cortisol, aspartate, valine, leucine and ornithine from the adrenal region, but the release appears to be from differing sources or cellular pools. Overall, the study revealed that both the adrenal region and caudal vein release a large number of amino acids and related substances. The caudal vein, and possibly other blood vessels as well, may be a major source of circulating dopamine.

Sulfate conjugates of catecholamines in the allantoic fluid of the chicken embryo.

In addition to free dopamine (DA), norepinephrine (NE), and epinephrine (E), the allantoic fluid of the 13-day-old chicken embryo contains sulfate conjugates of these three catecholamines (CAs). The concentration of DA sulfate is relatively low, while NE and E sulfates occur at levels similar to those of the free fractions. The comparatively low concentration of free CAs in the amniotic fluid, seen in a previous study, is confirmed. However, the amniotic barrier for sulfated CAs is much stronger, possibly absolute. Though some technical difficulties remain to be resolved, the chicken embryo may become a useful model for the study of the prenatal functions of CA conjugates.

Endogenous codeine: autocrine regulator of catecholamine release from chromaffin cells.

In addition to the catecholamines (CAs) dopamine (DA), norepinephrine (NE) and epinephrine (E), perifused chromaffin cells of the eel secrete codeine and morphine. In controls, the release of NE and E is strongly correlated, while there is no correlation with DA. Low, physiological concentrations of codeine (500 pg/ml) reduce the release of NE and E, while 8-fold higher concentrations stimulate an instant, transitory release of all three CAs. Much higher concentration of codeine (100 ng/ml), corresponding to the therapeutically effective range in the human, again reduce the release of NE and E. Physiological and very high concentrations of morphine have no clear effect on CA release, while an intermediate concentration (38 ng/ml) increases the secretion of all three CAs. The opiate antagonist naloxone lowers the basal CA secretion and prevents the morphine-induced CA increase. During naloxone perifusion, a normally non-effective concentration (40 ng/ml) of codeine reduces the CA release. It appears that codeine is an autocrine regulator which suppresses CA release via naloxone-insensitive receptors, and stimulates CA release via opiate receptor(s). Co-released morphine may modulate the action of codeine.

Release of conjugated catecholamines by the adrenal medulla equivalent of the American eel, Anguilla rostrata.

The in vivo perfusate of the cardiovascular system of the American eel (Anguilla rostrata) contains both free and conjugated fractions of dopamine, norepinephrine, and epinephrine. In vitro perifusion revealed that conjugated catecholamines are released from the adrenal medulla equivalent. Together with similar reports on mammals, this suggests that conjugated catecholamines are phylogenetically wide-spread components of the secretory cocktail of chromaffin cells. The present findings are compatible with an "active" role of the catecholamine conjugates.

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.

Circulatory catecholamines in the eel: origins and functions.

While the three catecholamines (CAs) dopamine (DA), norepinephrine (NE) and epinephrine (E), are wide-spread in tissues of the American eel (Anguilla rostrata), the bulk of these CAs in the systemic blood originates from chromaffin cells in the wall of the posterior cardinal veins. In addition, the brain and unidentified structures in the opisthonephric kidney also release appreciable quantities of CAs. The functional realms attributed to systematically circulating CAs in teleosts comprise cardiovascular, respiratory, osmoregulatory, metabolic and endocrinotropic actions. In the eel, cardiovascular and respiratory effects are well established. However, we were unable to prove a physiological role of the CAs in osmoregulation. On the other hand, the eel is the only species among five vertebrates of greatly varying phylogenetic position (the others: hagfish, lamprey, rat, human) in which physiological doses of E were hyperglycemic. As in lamprey and rat, DA and NE are released in the eel by physiological doses of E. In addition, DA and NE also release the respective other two CAs. The physiological significance of the catecholaminotropic (CA-tropic) interactions remains to be established; however, the CA-tropic effect of E does not require the presence of the brain or 'preganglionic' nerve cells. In the eel, mild stress causes an immediate 'unorthodox' drop of plasma CAs, while stronger stress is followed by the expected increase of plasma CAs.

Catecholaminotropic effects of catecholamines in a teleost fish, Anguilla rostrata.

Injections of physiological and supraphysiological doses of epinephrine (E) into cardiac-cannulated eels cause a dose-related increase of plasma dopamine (DA) and norepinephrine (NE) within 3 min. Likewise, both exogenous DA and NE increase the plasma titers of the respective other two catecholamines (CAs). The baseline titers of NE and E are closely correlated. Lack of a correlation of the baseline titers of NE and E with that of DA appears to be due to a faster disappearance rate of DA from the circulation. E is strongly hyperglycemic, and the weaker glycemic action of NE may be mediated via E release. The effects of E seem to depend on a spurt-like increase rather than its titer per se. The ability of the eel to cope with very fast, excessive increases of plasma CAs raises the question of the underlying mechanisms.

Effects of exogenous somatostatin and antisomatostatin on serum parameters of the American eel.

1. Bolus injections of a wide range of concns. of somatostatin and of antisomatostatin, in cardiac-cannulated eels had no specific effects on serum osmolality, sodium, potassium and chloride. 2. A presumably physiological dose of somatostatin had a pronounced and sustained hyperglycemic effect beginning 160 min after the injection, which was absent at higher doses. However, an extremely high dose caused an early, temporary hyperglycemia. 3. Antisomatostatin also caused a hyperglycemia, which appeared after 20 min and lasted less than 24 hr. 4. It appears that, in the freshwater eel, somatostatin affects both hyper- and hypoglycemic mechanisms and that these effects depend on its concn and/or site of action.

Catecholamines in head and body blood of eels and rats.

1. When compared with other vertebrates, the circulating titers of norepinephrine and epinephrine of the yellow eel are very low. 2. The ratio of the catecholamine titers in the eel differs from that reported for other vertebrates. 3. Following decapitation, the titers of the catecholamines are higher in head blood than in body blood of both unanesthetized and anesthetized eels. In decapitated rats, only the dopamine titer is higher in head blood. 4. As in the lamprey, agitation stress causes a drop of circulating catecholamines. However, other forms of stress cause the expected increase. 5. It appears that many data on catecholamines in both brain and circulation of vertebrates in general have been influenced by stress effects.