pp60v-src tyrosine kinase is expressed and active in sarcoma-free avian embryos microinjected with Rous sarcoma virus.

Early embryonic avian tissue is resistant to transformation by Rous sarcoma virus. To determine the nature of this resistance, we examined the expression and properties of the Rous sarcoma virus transforming protein pp60v-src, in infected embryonic chicken limbs in ovo. Lysates from Rous sarcoma virus-infected limbs contained the viral structural protein p19gag, as detected by immunoblot analysis, and showed pp60v-src kinase activity in vitro.

Nonclassical cannabinoid analgetics inhibit adenylate cyclase: development of a cannabinoid receptor model.

Extensive structure-activity relationship studies have demonstrated that specific requirements within the cannabinoid structure are necessary to produce potent analgesia. A three-point association between the agonist and the receptor mediating analgesia consists of: 1) the C ring hydroxyl, 2) the phenolic A ring hydroxyl, and 3) the A ring alkyl hydrophobic side chain. Potent tricyclic and bicyclic structures were synthesized as "nonclassical" cannabinoid analgetics that conform to this agonist-receptor three-point interaction model.

Regulation of adenylate cyclase by chronic exposure to cannabimimetic drugs.

Previous studies in this laboratory have demonstrated that a cloned neuroblastoma cell line (N18TG2) responds to delta 9-tetrahydrocannabinol (THC), the major psychoactive product of marihuana, with an attenuation of cyclic AMP accumulation that results from an inhibition of adenylate cyclase. The requirement for the Gi regulatory protein, stereoselectivity, pharmacologic specificity and cell selectivity of this response suggest that a receptor for cannabimimetic compounds may be associated with adenylate cyclase in the neuroblastoma cell. Presented here is a comprehensive investigation of cellular effects of chronic exposure to cannabimimetic agents.

Pertussis and cholera toxin ADP-ribosylation in Dictyostelium discoideum membranes.

We report a 39 kDa substrate for cholera and pertussis toxins is present in D. discoideum membranes. This protein did not co-migrate with alpha subunits of either Gs (45 kDa and 52 kDa) or Gi (41 kDa) from control mammalian cells.

Effects of treatment with N-methyl-N-nitrosourea, artificial sweeteners, and cyclophosphamide on adult rat urinary bladder in vitro.

The effect of N-methyl-N-nitrosourea (MNU), sodium saccharin, sodium cyclamate and cyclophosphamide on rat bladder explants in vitro was studied. MNU administered as a single dose or in multiple treatments induced concentration-dependent changes in urothelial ultrastructure and cell surface topography. In a single treatment protocol, extensive cytotoxicity was observed in both the urothelium and stroma at concentrations of 500 to 1000 micrograms/ml, establishing a toxic threshold within this range.

Tissue tropism and temporal expression of Rous sarcoma virus in embryonic avian limb in ovo.

Embryonic avian tissue is resistant to the transforming potential of Rous sarcoma virus (RSV) in ovo. Analysis of the pattern of host-viral interactions in the first semester of chick development has demonstrated that RSV is first expressed in a limited population of muscle precursor cells and proceeds to spread throughout the developing dorsal and ventral limb musculature. The number of non-muscle cells participating in the infection is initially low but gradually increases as development continues.

Ammonium sulfate modifies adenylate cyclase and the chemotactic receptor of Dictyostelium discoideum. Evidence for a G protein effect.

(NH4)2SO4 was found to activate adenylate cyclase in Dictyostelium discoideum membranes. The effect of (NH4)2SO4 on the enzyme was observed after pretreatment of membranes but could not be observed if the salt was added to the assay mixture. Activation was seen when membranes were pretreated with 0.

Cannabinoid inhibition of adenylate cyclase: relative activity of constituents and metabolites of marihuana.

delta 9Tetrahydrocannabinol (THC) has been shown to inhibit the activity of adenylate cyclase in the N18TG2 clone of murine neuroblastoma cells. The concentration of delta 9THC exhibiting half-maximal inhibition was 500 nM. delta 8Tetrahydrocannabinol was less active, and cannabinol was only partially active.

Thyroid effects on adenosine 3',5'-monophosphate levels and adenylate cyclase in cultured neuroblastoma cells.

Using neuroblastoma cells as a model of developing neurons, we have tested the hypothesis that thyroid hormones alter cAMP metabolism. Neuroblastoma cells were grown in serum-free defined medium for 48 h with or without thyroid hormones. Treatment with 20-200 nM 3,5,3'-triiodo-L-thyronine (T3) increased the accumulation of cAMP by intact cells without altering growth, gross morphology, or DNA or protein content.

Regulation of Dictyostelium discoideum adenylate cyclase by manganese and adenosine analogs.

Adenylate cyclase is the critical enzyme in the chemotactic signal relay mechanism of the slime mold amoeba, Dictyostelium discoideum. However, few studies examining the regulation of this enzyme have been performed in vitro due to the instability of enzyme activity in crude lysates. For studies presented in this communication, a membrane preparation has been isolated that exhibits a high specific activity adenylate cyclase that is stable during storage at -70 degrees C and under assay conditions at 27 degrees C.

Muscarinic pharmacology of the inhibition of adenylate cyclase in N18TG2 neuroblastoma cells.

This study characterizes the muscarinic cholinergic receptors associated with the inhibition of adenylate cyclase on N18TG2 neuroblastoma cell membranes. Agonists could be divided into two classes: oxotremorine, acetylcholine, carbachol and arecoline exerted the most efficacious and potent inhibition, while McN-A343, bethanechol and AHR-602 were partial agonists. Both quinuclidinyl benzilate and atropine maximally antagonized the inhibitory effect of McN-A343, carbachol and oxotremorine.

Cyclic nucleotide phosphodiesterase isozymes in neuroblastoma cells.

Adenosine 3',5'-cyclic monophosphate (cAMP) content of neurons is determined not only by the rate of synthesis but also by the rate of hydrolysis by cyclic nucleotide phosphodiesterases. Multiple forms of cyclic nucleotide phosphodiesterase exist in brain and other tissues, and these may be regulated by various hormones and neuromodulators. The present study examines this regulation in a cloned line of neuroblastoma cells (N18TG2).

Epithelial-stromal interactions in the adult bladder: urothelial growth, differentiation, and maturation on culture facsimiles of bladder stroma.

Analysis of the responsiveness of isolated adult urothelium to a series of different stromal cell-extracellular matrix combinations demonstrated the capacity of stromal cells to induce and maintain normal patterns of urothelial growth, differentiation, and maturation in vitro. By incorporating embryonic mesenchymal derived (Swiss 3T3) cells into type I collagen matrices, simplified three-dimensional tissue-like facsimiles of bladder stroma were derived. When recombined with sheets of isolated urothelium these facsimiles could approximately reproduce the capacity of natural stromal tissue to support the expression of normal urothelial tissue specific characteristics.

The tyrosine phosphorylation substrate p36 is developmentally regulated in embryonic avian limb and is induced in cell culture.

The 36-kD protein-tyrosine kinase substrate p36 has been variously postulated to be involved in membrane-cytoskeletal interactions, membrane traffic, and the regulation of phospholipase A2, and its phosphorylation may play some role in malignant transformation by avian sarcoma viruses. Because embryonic tissues are resistant to transformation by avian sarcoma viruses, we have examined the expression of p36 in the developing avian embryonic limb. The level of p36 increased progressively from day 5 to day 14 of development.

An assessment of the role of opioid receptors in the response to cannabimimetic drugs.

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin.

Involvement of Gi in the inhibition of adenylate cyclase by cannabimimetic drugs.

The cellular mechanism of action of the cannabimimetic drugs is examined using cultured cells. In membranes from N18TG2 neuroblastoma cells and the neuroblastoma X glioma hybrid cells, NG108-15, the psychoactive cannabinoid drugs and their nantradol analogs could inhibit adenylate cyclase activity. This response was not observed in either the soluble adenylate cyclase from rat sperm or membrane-bound adenylate cyclases from C6 glioma or S49 lymphoma cells.

Synthesis and release of vasoactive intestinal polypeptide (VIP) by mouse neuroblastoma cells: modulation by cyclic nucleotides and ascorbic acid.

The mouse neuroblastoma cell line N18TG2 synthesizes and secretes a VIP-like immunoreactive material. The majority of this VIP-like material from both cell and media extracts elutes on HPLC in the same position as porcine or rat VIP. Several additional peaks which appear in the media extracts may represent variant forms or degradation products of VIP.

Cannabinoid inhibition of adenylate cyclase. Biochemistry of the response in neuroblastoma cell membranes.

The inhibition of adenylate cyclase activity by cannabimimetic compounds in a membrane fraction from cultured neuroblastoma cells has been examined. The inhibition was shown to be concentration-dependent over a nanomolar range for both delta 9-tetrahydrocannabinol and its synthetic analog, desacetyllevonantradol. Inhibition was rapid and reversible.

An ultrastructural analysis of the physical organization of collagenous (type I) matrices: one determinant of urothelium maintenance in vitro.

Collagenous matrices, used as cell culture substrata, can be prepared from different collagen types in a variety of forms using a range of polymerization procedures. Type I collagen has been most frequently used either as dried collagen films or hydrated collagen gels. Sheets of isolated bladder urothelium, when plated onto such matrices prepared from type I collagen by different polymerization methods (eg.

Guanine nucleotide inhibition of adenylate cyclase in a membrane fraction from Dictyostelium discoideum.

The regulation of adenylate cyclase by guanine nucleotides was examined in a plasma membrane preparation from Dictyostelium discoideum. At concentrations of greater than 10 microM, GDP, GTP and a non-hydrolyzable GTP analogue, guanosine 5'-(beta-gamma-imino)triphosphate (Gpp(NH)p), inhibited the enzyme. Guanosine, GMP and ITP were ineffective.

Cannabinoid inhibition of adenylate cyclase. Pharmacology of the response in neuroblastoma cell membranes.

Adenylate cyclase in plasma membranes was inhibited by micromolar concentrations of delta 8-tetrahydrocannabinol and delta 9-tetrahydrocannabinol and by levonantradol and desacetyllevonantradol. This inhibition was noncompetitive for stimulation of the enzyme at the prostanoid receptor by prostaglandin E1 or prostacyclin, or at the peptide receptor by secretin or vasoactive intestinal peptide. Forskolin-activated adenylate cyclase was also inhibited by cannabimimetic agents.