ATP-Evoked Ca2+ Mobilisation and Prostanoid Release from Astrocytes: P2-Purinergic Receptors Linked to Phosphoinositide Hydrolysis

Astrocyte cultures prelabelled with either [3H]inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H-labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP-stimulated 3H-IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP-evoked 3H-IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP-induced phosphoinositide (PPI) breakdown and 45 Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of alpha 1-adrenoceptors also caused PPI breakdown and 45 Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H-IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP-stimulated TXA2 release was reduced. Our data provide evidence that functional P2-purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.     

Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A in Acinetobacter sp. Strain 4-CB1

4-Chlorobenzoate degradation in cell extracts of Acinetobacter sp. strain 4-CB1 occurs by initial synthesis of 4-chlorobenzoyl coenzyme A (4-chlorobenzoyl CoA) from 4-chlorobenzoate, CoA, and ATP. 4-Chlorobenzoyl CoA is dehalogenated to 4-hydroxybenzoyl CoA. Following the dehalogenation reaction, 4-hydroxybenzoyl CoA is hydrolyzed to 4-hydroxybenzoate and CoA. Possible roles for the CoA moiety in the dehalogenation reaction are discussed.     

Sarcolipin, the "proteolipid" of skeletal muscle sarcoplasmic reticulum, is a unique, amphipathic, 31-residue peptide.

The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid," i.e., a protein which is soluble in acidic CHCl3/CH3OH. We propose the name sarcolipin for this small protein, to signify its lipid-like solubility and association with the sarcoplasmic reticulum. We have determined the following amino acid sequence for sarcolipin, using protein chemistry methods: M E R S T R E L C L N F T V V L I T V I L I W L L V R S Y Q Y. This 31-residue sequence includes a 19-residue hydrophobic segment which probably spans the sarcoplasmic reticulum membrane. The molecular weight calculated from the sequence, 3733, agrees with that measured by fast atom bombardment mass spectrometry, showing that sarcolipin contains no attached fatty acyl or other prosthetic groups.     

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.     

Action of Endo-(1 → 6)-α-d-glucanases on the soluble dextrans produced by three extracellular α-d-glucosyltransferases of Streptococcus sobrinus

Four different α-d-glucosyltransferases (GTF) have been obtained from culture filtrates of Streptococcus sobrinus strains grown in the chemostat at pH 6·5 in complex medium supplemented with Tween 80. Three of the enzymes, GTF-S1, GTF-S3 and GTF-S4, converted sucrose into soluble glucans. Their limit of hydrolysis with endodextranase, the proportion of linear to branched oligosaccharides among the end products of enzymic degradation, and methylation analysis, all supported the view that the glucans were dextrans. The S1-dextrans were highly branched (32% of α-(1 → 3)-branch points), S3-dextrans were linear, and the branching of S4-dextrans was intermediate in value (9%). The enzymes that catalyze the synthesis of three such diverse dextrans were thus proved to be three different GTF, each with a characteristic specificity. Conditions of growth in the chemostat could be varied to provide maximum yields of either GTF-S1, -S3 or -S4.     

Differential effects of lithium on agonist-stimulated inositol polyphosphate formation in rat cerebral cortex slices: Selective actions on muscarinic cholinergic responses

Recent data indicate that BMY 7378 demonstrates high affinity, selectivity and low intrinsic activity at hippocampal 5-HT_1A receptors, suggesting that BMY 7378 may represent the first selective 5-HT_1A functional antagonist. The present study examined the agonist and antagonist properties of BMY 7378 at spinal cord 5-HT_1A receptors. In electrophysiological studies, iontophoretic administration of either the 5-HT_1A agonist 8-OH-DPAT (43.8 ± 5.4 nA) or BMY 7378 (46.3 ± 5.2 nA) significantly inhibited the firing rate of wide-dynamic-range dorsal horn units indicating that BMY 7378 demonstrates significant intrinsic activity at spinal cord 5-HT_1A receptors. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 ejection current (20–100 nA) which antagonized the 8-OH-DPAT induced inhibition of dorsal horn unit activity. In behavioral studies in the spinal rat, 8-OH-DPAT increased the animals' sensitivity to noxious levels of mechanical stimulation (ED₅₀ = 269 ± 24 nmol/kg) as did BMY 7378 (ED₅₀ = 295 ± 70 nmol/kg) with no statistically significant difference in the maximal response (Y_max) observed. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 dose (10–1100 nmol/kg) which blocked the hyperalgesic effect of 8-OH-DPAT, rather, each drug produced similar effects which were additive. Further, the 5-HT_1A agonist effects of BMY 7378 were blocked by the 5-HT_1A antagonist, spiperone. Therefore, both the electrophysiologic and reflex data indicate that BMY 7378 possesses significant intrinsic activity at spinal cord 5-HT_1A receptors, and like 8-OH-DPAT is a partial agonist at these receptors. Differences in BMY 7378 intrinsic activity at spinal cord as opposed to hippocampal 5-HT_1A receptors are discussed in terms of regional differences in G-proteins coupled to 5-HT_1A receptors in these two CNS regions.     

Identification of a serum-inducible messenger RNA (5B10) as the mouse homologue of calcyclin: tissue distribution and expression in metastatic, ras-transformed NIH 3T3 cells

A mouse mRNA, provisionally designated 5B10, has been cloned based on its inducibility by serum in quiescent murine fibroblasts. Here we report the full-length complementary DNA sequence and a partial characterization. There are about five copies of the gene in the mouse genome. Sequence analysis of the 5B10 coding region reveals 94 and 97% amino acid identity to human and rat calcyclin, respectively. Although the coding region has been highly conserved during evolution of the rodent and human genomes, the untranslated flanking sequences differ significantly. A protein of Mr about 8000 was produced by in vitro translation of the mRNA transcribed in vitro from 5B10 complementary DNA in a riboprobe vector. An antiserum raised against a portion of the predicted human calcyclin protein cross-reacted with this mouse protein. 5B10 mRNA was found in greatest amount in organs containing proliferating cells, e.g., epidermis, skin, stomach, uterus of pregnant mouse, placenta, and decidua. Brain, liver, mature thymus, and skeletal muscle had little or no detectable 5B10 mRNA. 5B10 mRNA levels were higher in cells treated with 7,12-dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate than in their normal counterparts, suggesting a role in tumorigenesis. In addition, high 5B10 mRNA levels were associated with metastatic ability in a series of ras-transformed cells, in proportion to levels of ras p21 expressed by the cells, implicating 5B10 even more deeply in carcinogenesis.     

Identification of phytochrome B amino acid residues mutated in three new phyB mutants of Arabidopsis thaliana

The growth and development of plants is regulated by light viathe action of photoreceptors which are responsive to the red/far-red,blue and UV regions of the spectrum. Phytochrome B (the apoproteinof which is encoded by the PHYB gene) is one of the red/far-redabsorbing photoreceptors active in this process. In this paper,the isolation and characterization of three new EMS-inducedmutations of Arabidopsis which confer phytochrome B deficiencyare described. Complementation analysis showed that these mutations(phyB-101, phyB-102 and phyB-104) were allelic with PHYB. DNAsequence analysis showed that all three mutants contain nucleotidesubstitutions in the PHYB-101 gene sequence. phyB-101 carriesa nucleotide substitution within the second exon of the PHYBgene. This G-to-A substitution is a missense mutation that convertsa glutamate residue at position 812 of the phytochrome B apoproteinto a lysine residue. phyB-102, another missense mutant, carriesa C-to-T substitution which converts a serine residue at position349 of the phytochrome B apoprotein to a phenylalanine residue.phyB-104 carries a premature stop codon as a result of a G-to-Amutation 1190 bp down-stream of the ATG start codon of the PHYBsequence. The missense mutations in phyB-101 and phyB-102 causesignificant alterations in the predicted second ary structureof their respective mutant polypeptides, and identify aminoacid residues playing crucial roles in phytochrome B function,assembly or stability. Key words: Arabidopsis thaliana, phytochromet, phyB mutants, missense mutations     

Dynamics of antarctic penguin populations in relation to inter-annual variability in sea ice distribution

To investigate the role of sea ice cover on penguin populations we used principal component analysis to compare population variables of Adélie (Pygoscelis adeliae) and chinstrap (Pygoscelis antarctica) penguins breeding on Signy Island, South Orkney Islands with local (from direct observations) and regional (from remote sensing data) sea ice variables. Throughout the study period, the Adélie penguin population size remained stable, whereas that of chinstrap penguins decreased slightly. For neither species were there significant relationships between population size and breeding success, except for an apparent inverse density-dependent relationship between the number of Adélie breeding pairs and the number of eggs hatching. For both species, no general relationship was found between either population size or breeding success and the local sea ice conditions. However, the regional sea ice extent at a particular time prior to the start of the breeding season was related to the number of birds that arrived to breed. For both species, this period occurred before the sea ice reached its maximum extent and was slightly earlier for Adélie than for chinstrap penguins. These results suggest that sea ice conditions outside the breeding season may play an important role in penguin population processes.