Chicken retinospheroids as developmental and pharmacological in vitro models: acetylcholinesterase is regulated by its own and by butyrylcholinesterase activity

Summary The phylo- and ontogenetically related enzymes butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are expressed consecutively at the onset of avian neuronal differentiation. In order to investigate their possible co-regulation, we have studied the effect of highly selective inhibitors on each of the cholinesterases with respect to their expression in rotary cultures of the retina (retinospheroids) and stationary cultures of the embryonic chick tectum. Adding the irreversible BChE inhibitor iso-OMPA to reaggregating retinal cells has only slight morphological effects and fully inhibits BChE expression. Unexpectedly, iso-OMPA also suppresses the expression of AChE to 35%–60% of its control activity. Histochemically, this inhibition is most pronounced in fibrous regions. The release of AChE into the media of both types of cultures is inhibited by iso-OMPA by more than 85%. Control experiments show that AChE suppression by the BChE inhibitor is only partially explainable by direct cross-inhibition of iso-OMPA on AChE. In contrast, the treatment of retinospheroids with the reversible AChE inhibitor BW284C51 first accelerates the expression of AChE and then leads to a rapid decay of the spheroids. After injection of BW284C51 into living embryos, we find that AChE is expressed prematurely in cells that normally express BChE. We conclude that the cellular expression of AChE is regulated by the amount of both active BChE and active AChE within neuronal tissues. Thus, direct interaction with classical cholinergic systems is indicated for the seemingly redundant BChE.     

Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.     

Determination of the enantiomeric purity of (−−) 2-(N-propyl-n-2-thienylethylamino)-5-hydroxytetralin (n-0923) by chiral stationary phase HPLC

The determination of the enantiomeric impurity, i.e., the percentage of (+) N?0437 (= N?0924) in several batches of (??) N-0437 (= N-0923) by chiral HPLC is described. Enantiomeric impurities were calculated based on the peak areas of the two baseline separated enantiomers in the chromatogram. The enantiomeric impurities found in different batches ranged from 0.02% to 0.11%. Calibration curves of the two isomers of N-0437 (Fig. 1,) were made twice to study the reproducibility and linearity of the method. The absorbance ratio, N-0923/N-0924, was found to be 1.02 with a relative standard deviation (RSD) of 9% over the whole concentration range used for the calibration curves.     

Different modes of electrogenic Na+ absorption in the coprodeum of the chicken embryo: role of extracellular Ca2+

Summary Transepithelial electrogenic Na⁺ transport (I_Na) was investigated in the coprodeum of 20-days-old chicken embryos in Ussing chambers. Short circuit current (I_sc) and transepithelial resistance (R_t) were 14.7±4.8 A · cm^-2 (n=12) and 0.53±0.09 k · cm^-2 (n=12), respectively. I_Na was calculated from changes in I_sc by substitution of mucosal Na⁺ by (N-methyl-d-glucamine) (NMDG). I_sc inversed during Na⁺ removal, and I_Na was found to be 27.8±4.7 A · cm^-2 (n=12). Amiloride (100 mol · l^-1) inhibited only about 60% of I_Na. Analysis of I_sc fluctuations revealed a Lorentzian component in the power density spectrum with a corner frequency of about 57 Hz. This component was not correlated to I_Na, and its origin is still unclear. Removal of mucosal Ca²⁺ increased I_Na about 2.5-fold due to an increase of the amiloride-insensitive component of I_Na in additionally investigated adult tissues. The results clearly show that this is due to a non-selective cation channel with an apparent order of selectivity Cs⁺>Na⁺=K⁺>Rb⁺>Li⁺. The Ca²⁺ concentration required to block 50% of the I_sc was about 18 mol · l^-1. The I _sc ^Ca could also be supressed by other divalent cations such as Mg²⁺ and Ba²⁺. Additionally, an I_Na-linked Lorentzian component occurred which dominated the control spectrum with a significantly higher corner frequency (about 88 Hz). The results indicate that Na⁺ absorption in the coprodeum of the chicken embryo is more complex than in adult hens. However, the Ca²⁺ sensitivity of I_Na is similar to comparable effects described for other epithelia. This possibly reflects the existence of two types of amiloride-insensitive apical cation channels as pathways for Na⁺ absorption, which may be involved to differing degrees in ontogenetic developments of nonselective channels to Na⁺-specific ion channels.Abbreviations DPL direct-linear-plot method - slope of the back-ground noise component - EGTA ethylene glycol-bi(2-amino-ethylether)-N,N,N,N-tetraacetic acid - f frequency - f _c corner frequency of the Lorentzian noise component - G _t transepithelial conductance - HEPES N-hydroxyethylpiperazine-N-ethanesulfonic acid - I _sc short-circuit current - I _Na transepithelial sodium current - I _sc ^Ca Ca²⁺-sensitive short-circuit current - K _m ^Ca Michaelis-Menten constant for Ca²⁺ - K _B power density of the background noise component at f=1Hz - m mucosal - NMDG N-methyl-D-glucamine - R _t transepithelial resistance - s serosal - SEM standard error of mean - S(f) power density of the Lorentzian noise component - S _o plateau value of the Lorentzian noise component     

Capacity of neural crest cells from various axial levels to participate in thymic development

Summary In order to assess the capacity of neural crest from different sources to participate in thymic development, neural crest from selected axial levels was transplanted unilaterally from quail donors to the region in chick hosts from which neural crest cells normaly migrate to interact with the primordial thymus. The greatest representation of donor cells was observed after isotopic transplantation and when donor tissue was taken from the hyoid and mesencephalic regions of the neural crest. The capacity for transplants to contribute cells decreased both anteriorly and posteriorly, so that neural crest close to the usual origin of mesenchyme-producing cells contributed a larger number of donor cells around the developing thymus than neural crest from anterior and posterior regions. Cells from the transplant were inserted as an addition to the host chick cells. Thus, a special relationship and capacity for interaction in thymic development is expressed by neural crest at usual levels over a limited span of axial regions, but to some extent by all regions. This study has established that the capacity for neural crest cells from different axial levels to interact with developing organs is not uniform, but may vary, depending upon the nature of the interaction with a particular organ.This study was supported by Grant No. 2332, The Council for Tobacco Research, USA, Inc.     

Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Effects of steroid hormones on the neuropil of the hypothalamic paraventricular nucleus of male chickens

Summary Testosterone and corticosterone, administered in doses of 0.5 mg/day for two weeks to three-day-old male chickens, induced alterations in the distributional pattern and in the number of synapses in the rostral neuropil of the hypothalamic paraventricular nucleus. This avian nucleus is a target area for both above-mentioned hormones and also one of the most important centers involved in the regulation of behavioral patterns related to reproduction. Testosterone increased the number of synapses in the rostral paraventricular nucleus, while corticosterone altered their distributional pattern causing an increase in type-B terminals; according to morphological criteria the latter are regarded to represent aminergic endings. Similar results were induced by simultaneous administration of both testosterone and corticosterone. Precocious sexual behavior was also provoked by double treatment.Preliminary results have been presented on the occasion of the 5th ENA meeting (Liège, Belgique, Sept. 1981) and the 1st Italian Meeting of Cell Biology (Rimini, Italy, April 1982)This study was supported by CNR bilateral grants (82.00215.04 & 83.00492.04), MPI 40% and European Training Program in Brain and Behaviour Researchs (twinning grant)     

Effect of initial developmental stage on morphology of transplanted embryonic chick pancreas

Summary It has been reported that only certain types of pancreatic parenchymal cells survive transplantation. This study examines whether the extent of differentiation of the pancreas at the time of transplantation affects the resulting morphology or viability of its components. Segments of chick pancreas or its primordia from stages preceding formation of dorsal bud (60 h) through hatching (day 21) were implanted in the abdominal region of three-day chick embryos. After various periods of growth, grafts were examined by light- and electron microscopy. In all transplants, individual endocrine cells (A, B, D, PP) and islet structure were identical to those of normal embryos of comparable age. The exocrine portion also appeared normal in implants from embryos younger than seven days. In grafts from older donors, however, normal acini and ducts were replaced by dilated, irregularly shaped tubules from which new islets appeared to develop. These results suggest that transplantation causes structural modification of exocrine tissue, which may reflect its initial functional capabilities. These observations are compared with similar structural alterations that occur following experimental obstruction of ducts and in human pancreatic pathology.     

Effects of chronic treatment with a GnRH agonist on oestrous behaviour and on the secretion of LH and progesterone in the ewe

A study was carried out to investigate a novel approach to oestrus synchronization in the ewe by treatment with a gonadotrophin releasing hormone (GnRH) agonist. Groups of ewes were initially treated on Day 2, 10 or 14 of the oestrous cycle with 10 mug GnRH analogue (D-Ser(Bu(t)) 6 des Gly GnRH ethylamide) per ewe per day for 14 days. Behavioural oestrus was inhibited during GnRH agonist treatment and recurred from 8 to 38 days after the treatment in an unsynchronized manner. Luteal activity during treatment was not impaired but reduced progesterone concentrations occurred in cycles after the treatment. The rhythm of ovarian function, generally characterized by prolonged follicular development, was impaired. During the treatment and subsequent recovery period, integrity of pituitary function was examined by measuring luteinizing hormone (LH) after GnRH agonist was injected, and after stimulation test doses of 150 ng natural GnRH were administered. During treatment there was, with time, a decline in pituitary response to the agonist which suggested that pituitary release of LH was exhausted. After the 14-day treatment the stimulation test with GnRH revealed a gradual return to normal responsiveness although this was not complete three weeks after the treatment when compared to control ewes. This lowered pituitary activity could cause the impaired ovarian function.     

Ontogenetic development of corticotropin-releasing factor (CRF)-containing neural elements in the brain of the chicken during incubation and after hatching

Summary In chicken embryos of different ages and in young chickens after hatching, neural elements reacting with antibodies generated against synthetic ovine corticotropin-releasing factor (CRF) were studied by means of the peroxidase-anti-peroxidase (PAP) technique at the lightmicroscopic level. CRF-immunoreactivity was first observed in perikarya located in the periventricular part of the hypothalamus on the 14th day of the incubation period. CRF-containing neural elements were detected on the same day of incubation in the external zone of the median eminence, but not in all investigated animals. In extrahypothalamic sites, immunoreactive perikarya were demonstrable in the central gray of the mesencephalon on the 15th day of incubation. Furthermore, immunoreactive cells appeared in other brain regions such as nucleus accumbens and dorsomedial nucleus of the thalamus after hatching. The present observations provide information regarding the functional development of the hypothalamo-hypophyseal-adrenal axis in the chick embryo.