Indole metabolism in the pineal organ of the pigeon with special reference to melatonin-synthesizing cells

By means of radioimmunoassay a clear-cut peak of melatonin concentration was found in the pineal organ of the pigeon at the middle of the scotophase (Coisin et al. 1982a). The aim of the present study was to identify the cell type responsible for the nocturnal indole metabolism, including melatonin synthesis, in the pineal of this avian species. After a short-term incubation or organ culture in the presence of [3H]-indolic precursors, [3H]-5-hydroxytryptophan or [3H]-5-hydroxytryptamine, the relative amounts of deaminated and acetylated products occurring in the pineal organ were measured by the use of thin layer chromatography and liquid-scintillation counting. It was possible to modify the relative amounts of deaminated and acetylated indoles by the application of some inhibitors of monoamine oxidase and cyclic nucleotide phosphodiesterase. Irrespective of the experimental conditions, high-resolution autoradiography combined with the above-mentioned radiochemical experiments showed that the cells of the receptor line (modified photoreceptor cells) are responsible for indole storage and metabolism, and very probably also for melatonin biosynthesis. The other cell types of the pineal parenchyma did not display significant labeling.     

Regulation of chicken pineal arylalkylamine-N-acetyl transferase by postsynaptic alpha 2-adrenergic receptors

The present investigation sought to characterize the adrenergic inhibition of arylalkylamine-N-acetyltransferase in cultured chicken pineal glands. Arylalkylamine-N-acetyltransferase, the melatonin rhythm generating enzyme, displays daily oscillations of activity that are driven by a circadian oscillator. Norepinephrine released at sympathetic nerve endings inhibits the enzyme and appears to play a role in maintaining a circadian rhythm of melatonin release. Chicken pineal glands were isolated in organ culture and the effects of adrenergic agents on the night time peak of N-acetyltransferase activity were studied. Norepinephrine and clonidine prevented 50 to 65% of the nocturnal rise of N-acetyltransferase activity. When applied at middark, norepinephrine and clonidine caused a 50 to 65% inhibition of N-acetyltransferase activity in 2 hours. Dose-response studies indicated clonidine was 100 times more potent than norepinephrine or cirazoline at inhibiting N-acetyltransferase activity. Inhibition of N-acetyltransferase activity was also observed, at micromolar concentration with epinephrine, UK 14,304 and alpha-methylnorepinephrine but not with phenylephrine, isoproterenol or dopamine. Epinephrine and clonidine actions were antagonized by yohimbine but not by prazosin. Destruction of the presynaptic compartment by bilateral superior cervical ganglionectomy did not affect the clonidine-induced inhibition of N-acetyltransferase and its reversal by yohimbine. It is concluded that the adrenergic inhibition of N-acetyltransferase activity in chicken pineal gland probably occurs via stimulation of postsynaptic alpha 2-adrenergic receptors.     

Plasticity of Astroglial Glutamate and γ-Aminobutyric Acid Uptake in Cell Cultures Derived from Postnatal Mouse Cerebellum

Abstract: The plasticity of astroglial glutamate and γ-aminobutyric acid (GABA) uptakes was investigated using mouse cerebellar cell cultures. The influence of external factors, such as different sera and/or the presence of neurons, was examined. Control autoradiography experiments showed that after short-term exposure to radioactive amino acids, granule cells took up neither glutamate nor GABA, and β-alanine predominantly inhibited astroglial GABA uptake. Astroglial uptake was quantified by measuring the radioactivity taken up by the cells in the culture and relating this measurement to the number of glial fibrillary acidic protein-positive cells present. Glutamate uptake was investigated in astroglial cultures and subcultures and in neuro-nal-astroglial cultures derived from postnatal day 4 mouse cerebella. In the absence of neurons, glutamate uptake increased during the first 9 days after plating and then leveled off. At 14 days in vitro in horse serum, which favors the differentiation of fibrous-like astrocytes, glutamate uptake related to astrocyte number was twice as high as in fetal calf serum. In the presence of cerebellar neurons, this rate was even higher. The specificity of the responsiveness of astrocytes to neurons with respect to glutamate uptake was investigated by comparing GABA uptake in the different culture conditions. Neurons also increased the rate of GABA uptake by astrocytes. Another component of the astroglial plasma membrane, the density of β-adrenergic receptors, was, however, not markedly affected by the presence of neurons. Hence, these results showed that in astrocytes plated from postnatal day 4 mouse cerebella, the level of neuro-transmitter uptake can be regulated in vitro by factors present in sera and by cerebellar neurons in the culture. However, this plasticity declined during development because astrocytes plated from postnatal day 8 cerebella and cultured under identical conditions were less active in glutamate uptake and were insensitive to the presence of horse serum. The latter observation suggested that the metabolic plasticity of astrocytes is restricted to a period defined early in cerebellar development and is no longer evident by postnatal day 8.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

H-2 antigens,on mast cell membrane,as target antigens for anaphylactic degranulation

When single mast cells were isolated by micromanipulation, specific H-2 antigen-bearing mast cells were degranulated upon incubation with alloimmune sera (DAAD). When specific alloantigens were presented by lymphoid cells only, no degranulation occurred. Only antigen-bearing mast cells were degranulated, irrespectively of the presence of antigen-bearing lymphoid cells. Therefore, in DAAD, anaphylactic alloantibodies can and must recognize specific H-2 antigens on the mast cell membrane and simultaneously deliver the degranulation signal, through an Fc-Fc receptor interaction on the surface of the same mast cell.     

Studies on autoimmune encephalomyelitis in the guinea pig. II. An in vitro investigation on the nature, properties, and specificity of the serum-demyelinating factor.

Complement-dependent demyelinating activity of whole brain homogenate (WBH)-induced experimental allergic encephalomyelitis (EAE) sera was tested on long term tissue cultures of in vitro myelinated fetal guinea pig cerebellum. Complement-fixing (CF) auto-antibodies were shown to be the responsible agents, as demonstrated in experiments where all reagents belonged to the same species: guinea pigs of outbred (Hartley) and even of inbred (S2 or S13) strains. These antibodies were of the IgG2 class as shown by Sephadex G-200 and DEAE cellulose fractionation experiments. The corresponding auto-antigen was present in the homogenate and myelin of the central nervous system (CNS) tissue. It was different from the encephalitogenic basic protein of CNS myelin (BP), as shown in experiments where the demyelinating auto-antibodies were induced, detected, and absorbed by WBH or by CNS myelin but not by BP. They were neither induced by nor cross-reacting with cerebroside and peripheral nervous system (PNS) tissue.