Asymmetric synthesis followed by transmembrane movement of phosphatidylethanolamine in rat liver endoplasmic reticulum

Studies with phospholipase C have indicated that two-thirds of the phosphatidylethanolamine of rat liver endoplasmic reticulum is located in the inner leaflet of the membrane bilayer. Phosphatidyl[¹⁴C]ethanolamine is synthesised in microsomes incubated with CDP[¹⁴C]ethanolamine. Using phospholipase C as a probe we have observed that the labelled phospholipid is initially (1–2 min) concentrated in the ‘outer leaflet’ of the membrane bilayer. The specific activity of this pool of phosphatidylethanolamine was 3.5 times that of the inner leaflet. If, however, the microsomes were opened with 0.4% taurocholate or the French pressure cell to make both sides of the bilayer available to phospholipase C, the phosphatidylethanolamine behaves as a single pool for hydrolysis. On longer incubation, up to 30 min, with CDP[¹⁴C]ethanolamine the specific activity of the outer leaflet phosphatidylethanolamine becomes close to that of the inner leaflet. In chase experiments, in which microsomal phosphatidylethanolamine was labelled by incubation with CDP[¹⁴C]ethanolamine for 1 min, the reaction stopped by addition of calcium, and the microsomes isolated by centrifugation and reincubated, labelled phosphatidylethanolamine was transferred from the ‘outer leaflet’ to the ‘inner leaflet’, so that both were equally labelled. These observations suggest that phosphatidylethanolamine is synthesised at the cytoplasmic leaflet of the endoplasmic reticulum and subsequently transferred across the membrane to the cisternal leaflet of the bilayer. Transmembrane movement is apparently temperature-dependent and independent of continued synthesis of phosphatidylethanolamine.     

Persistence of species obeying difference equations

The question of persistence of interacting species is one of the most important in theoretical ecology; when the system is governed by difference equations this question is particularly difficult to resolve because of the complicated dynamics of the model. The problem has usually been tackled via the concepts of asymptotic stability and global asymptotic stability, however the first is not a strong enough restriction since only orbits starting near a rest point are guaranteed to converge to the rest point, while the second as well as usually being extremely difficult to establish is surely too strong a condition, since it rules out for example a stable limit cycle. It is proposed here that a more biologically realistic criterion, and incidentally one which turns out to be more tractable, is that of cooperativeness, where all orbits are required eventually to enter and remain in a region at a non-zero distance from the boundary (corresponding to a zero value of at least one population). A theorem is proved giving conditions for cooperativeness, and is applied to some examples of predator-prey interactions, simple conditions on the parameters being obtained even for some ranges of the parameters where the dynamics are chaotic.     

Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG). The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance. AChRs were stained with fluroscent α-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells. These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.     

Bismuth uptake in rat testicular macrophages: a follow-up observation suggesting that bismuth alters interactions between testicular macrophages and Leydig cells.

Recent studies suggest that bismuth accumulates in Leydig cells. In addition, a reduced level of serum testosterone and a statistically significant reduction of Leydig cells have been observed. It was therefore hypothesized that Bi has a direct toxic effect on rat Leydig cells. We have now developed a method for double labeling of bismuth and ED-2 (a marker for testicular macrophages). The present data demonstrate that the heavily bismuth-loaded cells in rat testis, originally interpreted as being Leydig cells, are bismuth-loaded macrophages. Consequently, our data suggest a modified hypothesis regarding bismuth-induced interactions between testicular macrophages and Leydig cells.     

Changes in the concentration and size of testicular macrophages during development

Structural and functional interactions exist between Leydig cells and testicular macrophages of adult rats. Since the function of Leydig cells changes during critical periods of development and postnatal maturation, it is possible that macrophages are in part involved in regulating this process. As a first step towards gaining an understanding of the development of this paracrine phenomenon, I have undertaken a series of studies designed to determine when macrophages first become identifiable in the fetal tests and to determine whether the concentration or size of macrophages changes during important stages of testicular maturation. Macrophages were identified immunohistochemically in frozen sections of testis from rats at various prenatal and postnatal ages using commercially available monoclonal antibodies to proteins specific to rat macrophages. It was found that macrophages positive for these antigens were found only within the interstitial compartment and were commonly associated with clusters of presumptive Leydig cells that were negative for these antigens. Macrophages were first identifiable in the testis at Day 19 of fetal development. The number of macrophages/unit area of interstitium increased 15-fold between Day 20 of gestation and Day 47 postpartum. The cross-sectional area of the macrophages increased 1.7-fold between Days 13 and 47 postpartum. These results demonstrate that the number and size of testicular macrophages changes with age, suggesting a role for these cells during important times of testicular development and maturation.     

Development of cytoplasmic digitations between Leydig cells and testicular macrophages of the rat

Summary Testicular macrophages and Leydig cells from adult animals are known to be functionally coupled. For example, secreted products from macrophages stimulate testosterone secretion by Leydig cells. In adult rat testes, structural coupling also exists between these cells. This coupling consists of cytoplasmic projections from Leydig cells located within cytoplasmic invaginations of macrophages. Although macrophages are known to exist in the testis in immature animals, it is not known when these digitations develop. The purpose of the present study was to determine whether the time of their development coincides with known maturational events that occur in Leydig cells, particularly during the peripubertal period. Testes from rats at 20, 30 and 40-days-of-age as well as testes from mature rats weighing more than 500 gm were prepared for ultrastructural analysis. It was found that digitations form between 20 and 30-days-of-age. These structures varied from simple tubular projections to complicated branched structures, suggesting that digitations are more than simple invaginations of microvilli into coated vesicles as previously described. Subplasmalemmal linear densities were also observed within macrophages juxtaposed to Leydig cells. Collagen was commonly observed between macrophages and Leydig cells in animals 20 days old. These studies demonstrate that although macrophages are present in the testis in maximal numbers at 20 days-of-age, they do not form junctions with Leydig cells until day 30. This is just prior to the major increase in secretory activity of rat Leydig cells that occurs during puberty.     

Squalestatin 1, a potent inhibitor of squalene synthase, which lowers serum cholesterol in vivo.

Squalestatin 1 is a member of a novel family of fermentation products isolated from a previously unknown Phoma species (Coelomycetes). Squalestatin 1 is a potent, selective inhibitor of squalene synthase, a key enzyme in cholesterol biosynthesis; in vitro, 50% inhibition of enzyme activity is observed at a concentration of 12 +/- 5 nM (range of 4-22 nM). Squalestatin 1 inhibits cholesterol biosynthesis from [14C]acetate by isolated rat hepatocytes (50% inhibition at 39 nM) and by rat liver in vivo. In marmosets, a species with a lipoprotein profile similar to that of man, squalestatin 1 lowers serum cholesterol by up to 75%. This compound will allow further investigation of the control of the sterol biosynthesis pathway and could also lead to the development of new therapies for elevated serum cholesterol.     

Microsomal membranes contain phosphatidylcholine that equilibrates across the bilayer, and phosphatidylcholine that does not

Results of experiments using phosphatidylcholine transfer protein and phospholipase C as probes indicate that there are at least two pools of phosphatidylcholine in rat liver microsomes. One of these is preferentially labelled with [14C]choline and does not equilibrate across the bilayer. The second pool is labelled with [3H]glycerol and does equilibrate across the bilayer. Our observations also confirm that phosphatidylcholine exchange protein does not modify the distribution of phospholipids or cause randomization of the inner and outer leaflet pools of phosphatidylcholine when these are differentially labelled by [14C]choline.     

Biochemical consequences of follicle-stimulating hormone binding to testicular macrophages in culture

The present studies demonstrate that testicular macrophages respond to follicle-stimulating hormone (FSH) by: 1) stimulating the rate of incorporation of amino acids into secreted proteins; 2) increasing the rate of incorporation of uridine into RNA; and 3) enhancing the accumulation of intracellular cyclic adenosine monophosphate (cAMP; which was potentiated by the addition of 1 mM 3-isobutyl-1-methylxanthine; MIX). In addition, dibutyryl cAMP (dbcAMP) enhanced the incorporation of amino acids into secreted proteins; however, this cAMP analog had no effect on the incorporation of uridine into RNA. Finally, it was demonstrated that testicular macrophages possess specific receptors with a high affinity for FSH.     

Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.