Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.     

F.9 embryonal carcinoma cell calcitonin autocrine system: correlation between immunoreactive calcitonin secretion and calcitonin receptor number

Mouse teratocarcinoma cells in culture offer an in vitro system to study the initial steps of embryogenesis. It has been suggested that, at such early stages, cell functions are regulated by an autocrine process in which embryonic cells produce factors that in turn act on themselves. F.9 cells possess specific membrane receptors for calcitonin (CT) (120 fmol/mg of protein, Ka, = 3.5 X 10(8) M-1). These cells produce CT detected by heterologous radioimmunoassay in serum-free culture-conditioned medium (75 pg/10(7) cells/12 h). When F.9 cells are incubated in serum-free medium, CT binding and secretion concomitantly drop by 50% within the first 2 h, then increase progressively to an upper plateau after the sixth hour. Preincubation with 10(-6) M CT leads to disappearance of CT receptors and CT secretion in the culture medium up to 6 h. Avoiding accumulation of CT in the medium by a continuous flow rate for 6 h leads to a progressive decrease of CT receptors. In addition, retinoic acid treatment of cells induces a parallel progressive decrease of CT receptor number and of total CT synthesis. These results suggest a reciprocal regulation of CT receptors and CT secretion, or a close relationship between their regulations.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

The role of environmental sodium chloride relative to calcium in gill morphology of freshwater salmonid fish

Summary Ultrastructure, distribution and abundance of cell types were examined in the gills of two freshwater salmonid species, Salmo fario and Salmo gairdneri, in media of selected ion content. In plain hard water (PW) with high concentrations of Ca²⁺, Na⁺, and Cl^-, gill chloride cells (CC) were confined to trailing edges and interlamellar regions of filaments whereas in mountain soft water (MW) with low concentrations of Ca²⁺, Na⁺, and Cl^-, CC were more numerous on filaments and covered lamellae, particularly along trailing edges. CC also appeared on lamellae of PW trout acclimated to soft water in a pond. This proliferation was not alleviated when ambient Ca²⁺ levels were raised (MW + Ca²⁺) but regressed in elevated NaCl media (MW + NaCl). The regression process involved an initial covering of CC by pavement cells followed by cytolysis and then eventual disappearance of CC. In MW, mucous cells were distributed mainly on trailing edges and, to a lesser extent, leading edges of filaments; they were absent from lamellae regardless of external ion levels.The results of this study shed some light on the functional significance of CC in freshwater fish. It is suggested that proliferation of CC is an adaptive response to dilute freshwater (i.e. [NaCl]<0.1 mequiv·1^-1).     

Effects of glucagon on biosynthesis of the mitochondrial enzyme, carbamoyl-phosphate synthase I, in primary hepatocytes and Morris hepatoma 5123D

When freshly-dispersed rat hepatocytes are maintained in primary monolayer cultures, they quickly lose their capacity to synthesize the urea cycle enzyme, carbamoyl-phosphate synthase. The ability to synthesize many other proteins, e.g., serum proteins including albumin, is retained. After an initial recovery period following cell isolation (24-48 h), glucagon is able to restore the ability of cultured hepatocytes to make carbamoyl-phosphate synthase. mRNA encoding the enzyme is about 4-times higher in hepatocytes maintained for 48 h in the presence of glucagon compared to hepatocytes without the hormone, as judged by in vitro translational assays. The level of carbamoyl-phosphate synthase activity expressed in transformed hepatocytes is unique to each hepatoma. Here we show that Morris hepatoma 5123D has retained such expression, and actively synthesizes the enzyme when 5123D cells are placed in monolayer cultures. Unlike normal hepatocytes, however, synthesis continues uninterrupted at a high level whether or not glucagon is present. 5123D has higher levels of translatable carbamoyl-phosphate synthase mRNA than normal liver.     

Tissue uptake of circulating hyaluronic acid

Previous work in the rabbit has shown that there is a significant flux of plasma hyaluronic acid (HA) which is taken up and degraded mainly in the liver but also concentrated in the spleen. Purified 14C-labelled HA of high average molecular wt prepared by biosynthesis from D-[U-14C] glucose was injected i.v. in mice and its tissue distribution was determined by whole-body autoradiography during the next 24 h. As blood levels declined, radioactivity was concentrated in the liver and spleen as found in the rabbit, and also in bone marrow and lymph nodes. Distribution was uniform in liver tissue, concentrated and relatively persistent in the periphery of lymph nodes, and distinctly nodular within the spleen. Analysis of an aqueous liver extract taken 4 h after injection identified 14C in HA, in a macromolecular fraction resistant to fungal hyaluronidase, and in metabolites of low molecular wt. These findings confirm and extend observations based on tissue extraction in rabbits. The pattern of distribution through the body and the restricted localization within spleen and lymph nodes further suggest that HA is absorbed from plasma and tissue fluids by elements of the reticuloendothelial system.     

Differential size variations between transcriptionally active and inactive telomeres of Trypanosoma brucei

We have studied the genes coding for the variant-specific surface antigen (VSA) in a series of seven trypanosome clones derived from AnTat 1.1: 1.1 leads to 1.3 leads to 1.6 leads to 1.16 leads to 1.1C leads to 1.3B leads to 1.18 These genes are all telomeric (1-5), and their surrounding, although sometimes similar, differs in each case. The length between these antigen genes and the corresponding DNA end appears to increase at each antigenic switch, with however occasional sharp size reductions, often linked to the involvement of the telomere in gene expression. This increase is due to a constant "growth" of the telomeres, at a rate of about 28 bp per day in at least four cases and probably linked to chromosome duplication. The telomere harbouring the transcribed VSA gene is growing slightly faster (about 36 bp per day), and it is the only one whose size reduction is progressive, leading to a terminal length heterogeneity within a clone. As a result, the active VSA gene is found in a population of telomeres which, as the trypanosomes divide, becomes increasingly heterogeneous, with however a preferred discrete size class about 1.4 kb smaller. The fact that the "active" telomere is the only one in a chromatin conformation highly sensitive to DNAaseI (1-4, 6), suggests that chromatin structure influences the rate and extent of both size increase and shortening of telomeres.