sq-23377 and phorbol-12-13-didecanoate

In this study, we investigated the vasoactive intestinal polypeptide (VIP)-stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca2+ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4 beta-phorbol 12-myristate 13-acetate reduced VIP- but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E-115 cells, and that although activation of protein kinase C inhibits the VIP-stimulated cAMP response, elevation of intracellular Ca2+ potentiates this signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 1-Methyl-3-isobutylxanthine; Animals; Calcium; Carcinogens; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Drug Synergism; Enzyme Activation; Ionomycin; Isoquinolines; Kinetics; Mice; Neuroblastoma; Phorbol Esters; Piperazines; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Sphingosine-1-phosphate, a metabolite of sphingolipids which has previously been shown to stimulate DNA synthesis and cell division in quiescent cultures of Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167), induced a transient increase in intracellular free calcium independent of extracellular calcium. The increase in calcium was completely abolished when intracellular calcium pools were depleted with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. The dose-response for calcium release induced by sphingosine-1-phosphate correlated closely with the concentration required for stimulation of DNA synthesis. The magnitude of the calcium response decreased with successive challenges, although sphingosine-1-phosphate did not attenuate the responses to either bradykinin or ionomycin. Conversely, prior stimulation of the cells with bradykinin had no effect on the sphingosine-1-phosphate-induced calcium signal. Although sphingosine-1-phosphate increased inositol (1,4,5)-trisphosphate levels, complete inhibition of inositol phosphate formation by pretreatment with 12-O-tetradecanoylphorbol-13-acetate did not block sphingosine-1-phosphate-mediated calcium responses. Moreover, in permeabilized cells, heparin, an inositol (1,4,5)-trisphosphate antagonist, blocked Ca2+ release induced by inositol (1,4,5)-trisphosphate, but did not significantly alter the Ca2+ release induced by sphingosine-1-phosphate. Sphingosine-1-phosphate did not stimulate the release of arachidonic acid, another signaling molecule known to elevate [Ca2+]i without inositol lipid turnover or calcium influx. Our data suggest that sphingosine-1-phosphate mobilizes Ca2+ from internal stores primarily through a mechanism independent of inositol lipid hydrolysis and arachidonic acid release and that sphingolipid metabolism may be important in calcium homeostasis.

Topics: 3T3 Cells; Animals; Arachidonic Acid; Bradykinin; Calcium; Calcium-Transporting ATPases; Cytosol; DNA; Egtazic Acid; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionomycin; Kinetics; Lysophospholipids; Mice; Phorbol Esters; Second Messenger Systems; Sphingosine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Time Factors

Ionomycin, which mobilizes Ca2+, and phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), were used to compare the effects/interactions of Ca2+ and PKC on luteinizing hormone (LH) secretion from pituitaries of intact male and acutely ovariectomized (72 h) rats. Quartered pituitaries from donor animals were perifused at 0.25 ml/min and sequential effluent fractions were collected every 10 min. Continuous administration (4 h) of 1 nmol of gonadotropin-releasing hormone (GnRH) resulted in an increase in LH secretion. Cycloheximide (5 mumol) dissociated the GnRH-stimulated LH responses into protein synthesis-independent and -dependent components. While ionomycin (10 mumol) stimulated LH secretion from pituitaries of both sexes by protein synthesis-independent mechanisms, PMA (1 mumol) and the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (PDD), were ineffective secretagogues. However, PMA (but not PDD) interacted synergistically with ionomycin and GnRH to augment LH secretion by protein synthesis-dependent mechanisms. These results suggest a similarity in the effects/interactions of Ca2+ and PKC in gonadotropes from male and ovariectomized rats. If the effects of PMA can be attributed to PKC activation, then it also appears that Ca2+ mobilization is necessary for the manifestation of PKC as a mediator of LH secretion from these gonadotropes. While PKC activity can be divorced from the protein synthesis-independent component of LH release (this component appears to be mediated by Ca2+ mobilization), the enzyme might be involved in amplifying the response to Ca2+ mobilization through synergistic protein synthesis-dependent mechanisms.

Topics: Animals; Calcium; Cycloheximide; Female; Gonadotropin-Releasing Hormone; Ionomycin; Luteinizing Hormone; Male; Ovariectomy; Phorbol Esters; Pituitary Gland, Anterior; Protein Kinase C; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate

We have examined the effects of protein kinase C (PK-C) stimulation and cytosolic Ca2+ elevation on the in vitro induction of non-histocompatibility-restricted tumoricidal activity from human peripheral blood lymphocytes. The tumor cytolytic activity, as well as the number of cells recovered from interleukin 2 (IL-2)-stimulated cultures, was enhanced by the addition of the PK-C stimulator, phorbol dibutyrate (PDBu), but not non-PK-C-activating phorbol ester analogues while the Ca2+ ionophore, ionomycin, did not significantly alter development of IL-2-induced tumor cytolytic activity nor enhance cell yield. Neither PDBu nor ionomycin, alone or in combination, induced tumoricidal activity. The addition of both PDBu and ionomycin to recombinant interleukin 2 (rIL-2)-exposed cultures produced a strong mitogenic response and high cell yield, although Daudi cell killing measured at Day 5 was completely abolished. This abrogation of lymphokine-activated killer cell activity was seen as early as 24 h following exposure to PDBu and ionomycin, reaching 50% following 2 days of exposure. When lymphocytes mitogenically expanded by primary exposure to PDBu and ionomycin and then washed free of these agents were further cultured with rIL-2 alone, proliferation continued, and substantial cytolytic activity for Daudi cells was induced. The development of this postexpansion cytotoxic activity was not dependent on the addition of exogenous rIL-2 during the primary cultures. Fractionation of cells into large granular lymphocytes and small T-lymphocytes indicated that only the large granular lymphocytes proliferate in response to rIL-2 alone. Both large granular lymphocytes and small T-lymphocytes proliferate in response to the addition of PDBu and ionomycin, and both populations of cells developed tumor cytolytic activity following removal of PDBu and ionomycin and subsequent culture in rIL-2. These data suggest that PK-C and Ca2+ signals play key roles in the regulation and/or proliferation of tumor cytotoxic lymphocytes or their precursors and that manipulation of those signals can be utilized to produce substantially more tumoricidal activity from lymphocyte populations than can be achieved with rIL-2 alone.

Topics: Calcium; Cell Separation; Cytosol; Cytotoxicity, Immunologic; Ethers; Humans; Immunity, Cellular; In Vitro Techniques; Interleukin-2; Ionomycin; Killer Cells, Natural; Lymphocyte Activation; Phorbol Esters; Protein Kinase C; Time Factors