guanylyl-imidodiphosphate and mastoparan

Guanine nucleotide regulatory proteins (G-proteins) play an important role in the onset and progression of malignancy. We hypothesized that alterations in inhibitory G-protein (Gi) expression and/or function may contribute to cellular invasion and formation of hepatocellular carcinoma (HCC). H4IIE hepatoma cells were inoculated directly into the liver parenchyma of ACI strain rats, and membranes were prepared from HCC livers and adjacent nonneoplastic livers 12 days following the initial inoculation. Expression of inhibitory Gialpha proteins was determined by Western blot analysis and changes in the functional activity of these proteins confirmed by pertussis toxin catalyzed ADP ribosylation and adenylyl cyclase activity. Inhibitory Gialpha1, Gialpha1/2, and Gialpha3 protein expression was significantly elevated in HCC when compared to adjacent nonneoplastic liver and sham-operated hepatic tissue. Pertussis toxin catalyzed ADP ribosylation of Gialpha substrates was significantly enhanced in HCC concomitant with increased basal and stimulated adenylyl cyclase activity following uncoupling of Gi-proteins with manganese ions. The role of Gi-proteins in cellular proliferation was confirmed using cultured H4IIE cells and normal hepatocytes. In quiescent H4IIE cells, mastoparan (Gialpha activator) increased [3H] thymidine incorporation and cell growth in a dose-dependent manner, whereas both pertussis toxin (a Gi-protein inhibitor) and 8-bromo-cAMP inhibited mitogenesis. In contrast, in isolated cultured hepatocytes, mastoparan inhibited [3H] thymidine incorporation, while pertussis toxin and 8-bromo-cAMP were mitogenic. We conclude that HCC is associated with marked changes in Gialpha-protein expression in vivo and in vitro, direct activation of which leads to increased mitogenesis in H4IIE cells in vitro.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Carcinoma, Hepatocellular; Cell Division; Cells, Cultured; Chlorides; Cholera Toxin; DNA; DNA, Neoplasm; GTP-Binding Protein alpha Subunits, Gi-Go; Guanylyl Imidodiphosphate; Intercellular Signaling Peptides and Proteins; Liver; Liver Neoplasms, Experimental; Male; Manganese Compounds; Peptides; Pertussis Toxin; Poly(ADP-ribose) Polymerases; Rats; Rats, Inbred ACI; Tumor Cells, Cultured; Virulence Factors, Bordetella; Wasp Venoms

Regulated exocytosis requires both calcium and MgATP. Although the biochemical events responsible for ATP-dependent calcium-activated secretion have not been elucidated yet, some progress has been made in determining the relative order of the ATP- and calcium-dependent steps. Studies on permeabilized secretory cells have shown that MgATP acts before calcium and maintains the secretory apparatus in a "primed" state. In this paper, we examine the possible role of heterotrimeric G-proteins in these two steps of exocytosis in permeabilized chromaffin cells. We show that mastoparan and other activators of heterotrimeric G-proteins inhibit the MgATP-dependent reaction, but stimulate the late calcium-dependent step of exocytosis. Non-hydrolyzable GTP analogues (GTP-gamma-S and GMP-PNP) mimic the dual effects of mastoparan on secretion, but with different potencies, suggesting the involvement of two distinct heterotrimeric G-proteins in regulated exocytosis. GPAnt-2, a substance P related peptide known to inhibit the stimulation of Gi and Go by mastoparan, reverses, in a dose-dependent manner, both the inhibitory and stimulatory effects of mastoparan on secretion. These results indicate that two distinct heterotrimeric G-proteins from the Gi/o family may act in series in the exocytotic pathway in chromaffin cells: one controls the ATP-dependent priming step, whereas the second is involved in the late calcium-dependent fusion step which does not require ATP.

Topics: Adenosine Triphosphate; Aluminum Compounds; Amino Acid Sequence; Animals; Calcium; Cattle; Chromaffin System; Exocytosis; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanylyl Imidodiphosphate; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Norepinephrine; Oligopeptides; Peptides; Protein Conformation; Pyrrolidonecarboxylic Acid; Substance P; Wasp Venoms

Effects of guanine nucleotides on glucose transport were studied in permeabilized rat epididymal fat cells. GTP gamma S and Gpp(NH)p, but not App(NH)p, stimulated 3-O-methylglucose transport. Effect of GTP gamma S was dose-dependent, being detectable at 0.1 mM, and 1.0 mM GTP gamma S stimulated glucose transport to the same extent as insulin. GTP gamma S (0.3 mM) enhanced insulin-stimulated glucose transport while 1 mM GTP gamma S did not affect insulin-mediated transport. GDP beta S had no effect on glucose transport by itself but rather enhanced insulin action. NaF, which is known to activate trimeric G proteins, increased glucose transport to the same extent as insulin. Likewise, mastoparan augmented glucose transport. These results indicate that a certain type of trimeric G protein(s) is involved in the regulation of glucose transport.

Topics: 3-O-Methylglucose; Adenylyl Imidodiphosphate; Adipose Tissue; Animals; Biological Transport; Cell Membrane Permeability; Cells, Cultured; Dose-Response Relationship, Drug; Electric Stimulation; Glucose; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanylyl Imidodiphosphate; Insulin; Intercellular Signaling Peptides and Proteins; Male; Methylglucosides; Peptides; Rats; Rats, Sprague-Dawley; Sodium Fluoride; Thionucleotides; Wasp Venoms