P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol trisphosphate in rat hepatocytes

Stimulation of P2-purinergic receptors by ATP resulted in activation of phosphorylase, which was associated with marked production of inositol trisphosphate (Ins-P3), in rat hepatocytes. ATP also inhibited forskolin-induced accumulation of cAMP in the presence of a phosphodiesterase inhibitor. On the contrary, adenosine or AMP never inhibited the cAMP accumulation, but increased hepatocyte cAMP; the stimulation was antagonized by a methylxanthine. Thus, P1-purinergic receptors are linked to adenylate cyclase in a stimulatory fashion in hepatocytes. Various kinds of purine nucleotides stimulating P2-receptors can be divided into two groups on the basis of their relative abilities to stimulate Ins-P3 production and to inhibit cAMP accumulation; the first group including adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, 5-adenylyl imidodiphosphate, GTP, and guanosine 5'-O-(3-thiotriphosphate) has an efficacy similar to that of ATP, and the second group of nucleotides including alpha, beta-methyleneadenosine 5'-triphosphate, beta, gamma-methyleneadenosine 5'-triphosphate (App(CH)2)p), and GDP exerts considerable inhibitory effects on cAMP accumulation, but only slight effects on inositol lipid metabolism. Treatment of hepatocytes with islet-activating protein, pertussis toxin, blocked the nucleotide-induced inhibition of cAMP accumulation, but exerted only a small effect on Ins-P3 production. In membranes prepared from hepatocytes, forskolin-stimulated adenylate cyclase was inhibited by GTP. This GTP-induced inhibition of the enzyme was susceptible to islet-activating protein and dependent on the concentration of ATP (or its derivatives, ATP gamma S or App(CH2)p). It is concluded that there are two types of P2-purinergic receptors: one is linked to adenylate cyclase via an inhibitory guanine nucleotide regulatory protein (Gi) and the other is linked to phospholipase C.     

A new GTP-binding protein in brain tissues serving as the specific substrate of islet-activating protein, pertussis toxin

A new GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was purified from porcine brain membranes as an alpha beta gamma-heterotrimeric structure. The alpha-subunit of the purified protein (alpha 40 beta gamma) had a molecular mass of 40 kDa and differed from that of Gi (alpha 41 beta gamma) or Go (alpha 39 beta gamma) previously purified from brain tissues. The fragmentation patterns of limited tryptic digestion and immunological cross-reactivities among the three alpha were different from one another. However, the beta gamma-subunit resolved from the three IAP substrates similarly inhibited a membrane-bound adenylate cyclase and their beta-subunits were immunologically indistinguishable from one another. Thus, the alpha 40 beta gamma is a new IAP substrate protein different from Gi or Go, in the alpha-subunit only.     

Stellate cells storing retinol in the liver of adult lamprey,Lampetra japonica

Summary Distribution, localization and fine structure of the stellate cells in the liver of lamprey, Lampetra japonica, were studied during the spawning migration by use of Kupffer's gold-chloride method, fluorescence microscopy for vitamin A (retinol) and electron microscopy. The stellate cells in the lamprey liver differ in some of their properties from those in mammalian livers. Stellate cells which store abundant retinol in lipid droplets, occur not only in the hepatic parenchyma, but also in the dense perivascular and capsular connective tissue of the liver and in the interstitium of pancreatic tissue. In the hepatic parenchyma these cells are located perisinusoidally or along thick bundles of collagen fibrils. The stellate cells display a number of large retinol-containing lipid droplets, granular endoplasmic reticulum, tubular structures, dense bodies, Golgi complex, microtubules, and microfilaments. In the space of Disse, the stellate cells and extracellular fibrilar components such as collagen fibrils and microfibrils (11–12 nm in diameter) are intervened between the two layers of basal laminae. Differentiation and possible functions of the stellate cells in the lamprey liver are discussed.     

Splenic outer periarterial lymphoid sheath (PALS): An immunoproliferative microenvironment constituted by antigen-laden marginal metallophils and ED2-positive macrophages in the rat

Summary In an attempt to reveal the role of antigen-laden marginal metallophil (MM) and other macrophages in the intrasplenic immune response of a specific B-cell lineage to a thymus-independent type-2 antigen (Ficoll conjugated with fluorescein isothiocyanate), simultaneous immuno-histological observations of the involved cells were performed in the rat. By newly established methods of double or triple immunostainings, time-kinetics of the following parameters were studied and compared: (1) the antigen, (2) the specific antibody-forming cells (AFC) directed to the fluorescein-isothiocyanate determinant, (3) proliferating cells labeled with 5-bromo-2-deoxyuridine (BrdU), and (4) macrophage subpopulations recognized by monoclonal antibodies (ED2 and ED3). The antigen localized stably not only in the marginal-zone macrophages but also in the MM except around the follicular area. The increase of BrdU-positive cells was observed from day 2 up to day 4 after antigen injection mostly in the periphery of the periarterial lymphoid sheath (outer PALS), which indicated antigen-induced proliferation. As a novel finding, the majority of AFC, both BrdU-positive and -negative, were either closely associated with the antigen-laden MM, or forming cell clusters with ED2-positive macrophages in the outer PALS. In contrast, there were very few AFC in juxtaposition to antigen-free MM in the follicular area or the antigen-laden marginal zone macrophages. The results led to the proposal of a hypothesis that the antigen-laden MM together with ED2-positive macrophages constitute an immunoproliferative microenvironment for the plasmacellular reaction by accumulating the antigen-specific B-cell lineage and promoting these cells to differentiate into the AFC and to proliferate in the outer PALS.Abbreviations AFC specific antibody-forming cells - BrdU 5-bromo-2-deoxyuridine - Fic-F FITC-conjugated Ficoll - FITC fluorescein isothiocyanate - HRP horseradish peroxidase - MM marginal metallophils - MZ marginal zone - PALS periarterial lymphoid sheath - PBS phosphate-buffered saline - TI2 thymus-independent type-2     

Purification of GTP-binding proteins from bovine brain membranes. Identification of heterogeneity of the alpha-subunit of Go proteins

Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with alpha beta gamma-subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as alpha-subunit alone and was also ADP-ribosylated by IAP in the presence of beta gamma-subunits. When each alpha-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as Gi-1 and Gi-2, and other 4 appeared to be Go or Go-like G proteins. The alpha-subunits of immunologically Go-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the alpha-subunit of Go in the brain membranes.     

A pertussis toxin-sensitive GTP-binding protein plays a role in the G0-G1 transition of rat hepatocytes following establishment in primary culture

Acute spontaneous c-myc gene expression and sustained increase of a GTP-binding protein(s) (G-protein) which is sensitive to islet-activating protein (IAP), pertussis toxin, occurred early during primary culture of adult rat hepatocytes. Following these earlier events, DNA synthesis was demonstrated in response to EGF and insulin. Addition of IAP immediately after plating of primary cultures inhibited c-myc expression and the hormone-induced DNA synthesis. Addition at 24 h or later following cell inoculation, however, produced only weak effects on DNA synthesis, even though the IAP-sensitive G-proteins were completely inactivated. We conclude that the IAP-sensitive G-protein(s) plays a role in the earlier process(es) of the G0-G1 transition, which is essential for the initiation of growth factor-dependent DNA synthesis.     

Three-dimensional structure of endothelial cells in hepatic sinusoids of the rat as revealed by the Golgi method

Summary The three-dimensional structure of endothelial cells in the hepatic sinusoids of the rat was studied by application of light- and electron microscopy on Golgi-impregnated specimens. A number of endothelial cells could thus be individually delineated throughout the hepatic lobules. The cytoplasm, showing heavy silver deposits, consists of two distinct areas, a thick and thin portion. The thick portion, issuing from the region of the perikaryon, branches and tapers toward the cell periphery. The thin portion, occupying the remainder of the cytoplasm, consists largely of highly fenestrated sieve plates. Some intralobular variation can be noted; the thick portion of the endothelial cells is well developed in the periportal zone, while the cells in the centrilobular zone are relatively rich in thin portions. In addition, the area of distribution of an individual endothelial cell is larger in the centrilobular sinusoids than in the periportal zone. Some endothelial cells also possess unique cytoplasmic processes projecting into the intercellular space between hepatocytes and connecting the sinusoidal walls of neighboring sinusoids. These processes may anchor the endothelial cells to the hepatic plates.     

Induction of refractoriness of cyclic AMP responses to prostaglandin E1 and epinephrine by prior exposure of guinea pig macrophages to lipopolysaccharide

Prior exposure of guinea pig macrophages to LPS (lipopolysaccharide) resulted in reduced cAMP-generating responses to prostaglandin E1 and epinephrine. LPS-induced refractoriness was diminished when LPS treatment was carried out in the presence of an inhibitor of prostaglandin synthesis, hydrocortisone, or indomethacin, or an inhibitor of protein synthesis, cycloheximide. The release of arachidonic acid and its metabolites, especially prostaglandin E2 and thromboxane B2, increased during incubation of macrophages with LPS. These increases were efficiently antagonized by hydrocortisone, indomethacin, or cycloheximide. Preincubation of macrophages with prostaglandin E1 greatly reduced the subsequent responses of cAMP generation to prostaglandin E1 and unexpectedly also to epinephrine. Thus, increased production of prostaglandins during the LPS treatment is likely to be responsible for decreased cAMP responses to subsequent addition of prostaglandin E1 and epinephrine.     

Coupling of the guanine nucleotide regulatory protein to chemotactic peptide receptors in neutrophil membranes and its uncoupling by islet-activating protein, pertussis toxin. A possible role of the toxin substrate in Ca2+-mobilizing receptor-mediated signal transduction

A chemotactic peptide stimulated the high-affinity GTPase activity in membrane preparations from guinea pig neutrophils. The enzyme stimulation was inhibited by prior exposure of the membrane-donor cells to islet-activating protein (IAP), pertussis toxin, or by direct incubation of the membrane preparations with its A-protomer (the active peptide) in the presence of NAD. The affinity for the chemotactic peptide binding to its receptors was lowered by guanyl-5'-yl beta, gamma-imidodiphosphate (Gpp(NH)p) reflecting its coupling to the guanine nucleotide regulatory protein in neutrophils. The affinity in the absence of Gpp(NH)p was lower, but the affinity in its presence was not, in the A-protomer-treated membranes than in nontreated membranes. The inhibitory guanine nucleotide regulatory protein of adenylate cyclase (Ni) was purified from rat brain, and reconstituted into the membranes from IAP-treated cells. The reconstitution was very effective in increasing formyl-Met-Leu-Phe-dependent GTPase activity and increasing the chemotactic peptide binding to membranes due to affinity increase. The half-maximal concentration of IAP to inhibit GTPase activity was comparable to that of the toxin to inhibit the cellular arachidonate-releasing response which was well correlated with ADP-ribosylation of a membrane Mr = 41,000 protein (Okajima, F., and Ui, M. (1984) J. Biol. Chem. 259, 13863-13871). It is proposed that the IAP substrate, Ni, couples to the chemotactic peptide receptor and mediates arachidonate-releasing responses in neutrophils, as it mediates adenylate cyclase inhibition in many other cell types.     

Cell lines from imaginal discs ofDrosophila melanogaster

Summary New cell lines, designated as ML-DmDl≈10, were established from dissociated imaginal discs ofDrosophila melanogaster. The culture medium was prepared by mixing in a 1:1 ratio Cross and Sang’s M3(BF) medium, supplemented with 10% heat inactivated fetal bovine serum (FBS), with the supernatant of a primary embryonic cell culture made in the M3(BF) medium and supplementing this mixture with insulin. One cell line was established in the medium containing larval hemolymph instead of the primary culture supernatan, and another was established in fresh M3(BF) medium supplemented with insulin and FBS. In these mediums, imaginal disc cells first formed aggregates and cellular vesicles within a few weeks followed by the proliferation of thin-layered cells around them after about 1 mo. Ten cell lines have so far been established from two kinds of imaginal discs and disc mixtures. The ploidy of these cell lines was predominantly diploid. Population doubling time was about 50 to 70 h at 3 to 10 mo. after initiation of the culture. When the cell aggregates formed in vitro were implanted in metamorphosing larvae, they differentiated at high frequency into adult cuticular strutures in the early phase of the primary culture. This differentiation of aggregates was also observed, though at low frequency, in a culture maintained by dilution-transfer for 6 to 15 mo. in vitro.