Protein kinase C isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from mouse pancreatic islets

Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assess the PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulin release (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 mM) enhanced PIR ~ 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by ~ 40-50 percent. Both effects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cch also evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronic TMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. In contrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-induced insulin secretion.

Pretreatment with phorbol ester and an LHRH asgonist reduces testosterone production and protein kinase C activity in rat Leydig cells challenged with PDBu and LHRH

We have investigated the role of protein kinase C (PK-C) in luteinizing hormone-releasing hormone (LHRH)-induced testosterone secretion from purified rat Leydig cells (70-80-day old Sprague-Dawley rats) by pretreating the cells in vitro with 200 mM phorbol 12,13-dibutyrate (PDBu) (a known procedure to down-modulate this enzyme in most cell types) and 1 muM [D-Ala6,Des-Glyl0]-LHRH ethylamide, an LHRH agonist (LHRH-A). Following pretreatment we measured PK-C activity and secretion of testosterone in response to subsequent challenges with the PK-C activator PDBu (20-2000 nM) and with LHRH (0.001-1.0 muM) and the Ca2+ mobilizing secretagogue A23187 (0.1-1OO muM) in the same cell preparation. PDBu and LHRH-A pretreatments caused a reduction in testosterone secretion in response to subsequent exposure to PDBu or LHRH. Both pretreatments decreased PK-C activity in crude and purified extracts of the same cells. The magnitude of reduction of the secretory response was greater than that of enzyme activity for both PDBu and LHRH-A pretreatment (68.9 per cent reduction of testosterone secretion vs 54.7 per cent reduction of PK-C activity in PDBu-pretreated cells and 78.6 per cent reduction of testosterone production vs 36.6 per cent reduction of PK-C activity in LHRH-A-pretreated cells). The effect of phorbol ester pretreatment on PDBu- or LHRH-stimulated testosterone secretion and PK-C activity was specific (no measurable effect with 4 alpha-PDBu, an inactive phorbol ester). While PDBu and LHRH-A pretreatment reduced Leydig cell responsiveness to PDBu or LHRH, the secretion of testosterone in response to the Ca2+ -mobilizing secretagogue A23187 was similar in PDBu- and LHRH-A-pretreated and in control (non-pretreated) cells. We conclude that down-modulation of protein kinase C by prolonged exposure of Leydig cells to phorbol esters or LHRH-A results in decreased PK-C activity and testosterone secretion. These results provide the first evidence that pretreatment with LHRH-A, which does not enter the cell, can affect the steroidogenesis and PK-C activity responses to PDBu (the intracellular ligand of PK-C).

Slow cluster formation of purified human or rhesus T cells requires protein kinasse C and LFA-1

Ab: Homotropic T cell adhesion, as generally studied, consists of a rapid, transient binding process that is measured over a 15-120 min. period. Here we report a slow type of adhesion process occurring with human or rhesus T cells, purified from peripheral blood, that manifests itself by the formation of rounded, multi-layer clusters which may contain hundreds of cells. The maximal number and size of the clusters peak 1-2 days after the addition of phorbol ester, an absolute requirement. The number of clusters formed is proportional to phorbol ester concentration up to 1.25 ng/mL. Phorbol esters such as phorbol myristate acetate (PMA), phorbol dibutyrate (PDB), and 7-octylindolactam (OIL) induced optimal cluster formation at 1-13 ng/mL, levels slightly higher than that required to induce mitogenesis of purified T cells. Phorbol itself and the alpha-form of the ester were inactive. Both cluster formation and mitogenesis (stimulated by Con A or anti-CD3) are completely inhibited by staurosporin at 12.5 ng/mL. Even at 2.5 ng/mL, 74 percent of cluster formation was inhibited, which strongly implies a crucial role for protein kinase C. In the presence of accessory cells, T cell clusters were suppressed. Monoclonal Ab such as anti-CD3, mouse anti-CD3 followed by anti-mouse IgG, anti-CD4, anti-CD4A, anti-CD2, anti-CD8, and anti-CD45 did not induce cluster formation. None were inhibitory or stimulatory in the presence of PMA, except for anti-CD3 which enhanced cluster formation by 26 percent. However, anti-LFA-1 beta-chain (mouse monoclonal) completely blocked cluster formation over the range studied (63-1000 ng/mL) for both human and rhesus cells; rat anti-LFA-1 only blocked human cell adhesion. Anti LFA-1 only partially inhibited T cell mitogenesis. These results show that slow cluster formation shares the LFA-1 and phorbol ester requirements of the rapid adhesion of T cells requiring LFA-1 and ICAM-1. However, cluster occurs at a very low phorbol ester concentration, appears more sensitive to staurosporin inhibition, and is not stimulated via the TCR receptor like the rapid adhesion process. We hypothesize that certain neuronal processes, induced by phorbol ester, and which also show a similar protein kinase C activation time course, may share mechanisms in common with cluster formation

Inhibitory action of in vitro ethanol and acetaldehyde exposure on LHRH and phorbol ester stimulated testerone secretion by rat testicular interstitial cells

Ha sido demostrado que el etanol y el acetaldehido inhiben la esteroidogenesis testicular. No obstante el (los) mecanismo(s) de trasnducción de señal envuelto en su acción no fue aún elucidado. Hemos examinado el posible envolvimiento de la proteína quinasa sensible a fosfolípidos y dependiente de calcio (proteína quinasa C, PK-C) en el mecanismo intracelular de acción de etanol y de acetaldehido, estimulando la producción de testosterona en células intersticiales de testículo de rata con LHRH y éster de forbolPDBu, los cuales activan la PK-C a nivel de receptor (LHRH) y pos-receptor (PDBu). El etanol(2000 mg por ciento) inhibió la producción de testosterona estimulada con 10**-7 M LHRH y 200 nM PDBu en 81 + 4,7 por ciento y 60 + 20.4 por ciento respectivamente. El acetaldehido (20 mg por ciento) redujo la cantidad de testosterona producida por 10**-7 M LHRH y 200 nM PDBu en 59,4 + 1,2 por ciento, respectivamente. El nivel basal de testosterona no fue modificado por el etanol pero si fue reducido por el acetaldehido. No obstante, la prueba funcional de la viabilidad celular a través de la preincubación de las células con las referidas dosis de etanol y acetaldehido no disminuyó la capacidad de respuesta a una subsecuente estimulación con LHRH, demostrando que la viabilidad celular no fue afectada por la incubación con esas substancias. Los resultados aquí presentados sugieren que la exposición directa al etanol y acetaldehido redujo la habilidad de las células intersticiales del testículo de rata de responder a la estimulación de la vía mediada por la PK-C

Correlation between tissue growth kinetics and modulation of mouse skin tumorigenesis by phorbol esters.

Previous studies on the influence of phorbol esters on mouse skin tumorigenesis have shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances development of malignant epithelial and mesenchymal skin tumors by a completely carcinogenic dose of 3-methylcholanthrene (MCA), while its congener phorbol-12, 13-diacetate (PDA) exerts an inhibitory effect. Differential effects of these two agents were analysed by histology, morphometry and cell kinetic techniques including autoradiography and estimation of labelled precursor incorporation into DNA by liquid scintillation counting. Epidermal hyperplasia induced on exposure of S/RV Cri mouse skin to a single or multiple TPA application after MCA injection was associated with a significant increase in the thickness of nucleated cell layers, stratum granulosum, number of suprabasal cells and dark basal cells. Enhancing effect of TPA on MCA-induced neoplastic development correlated well with an increase in mitotic activity, number of cells in S-phase and increased rate of DNA synthesis in the epidermis, dermis and subcutis as also mast cell number. In contrast, treatment of MCA-injected preneoplastic mouse skin with PDA resulted in epidermal hypoplasia and cellular damage evident as cytoplasmic vacuolation and nuclear pyknosis. Multiple PDA exposure also reduced the thickness, mitotic index and number of cells in S-phase in epidermis, dermis and subcutis. Thus, cellular toxicity and inability to recruit cells in DNA-synthetic phase may account for inhibition of progression of preneoplastic epithelial and mesenchymal cells into overt tumors by PDA.

Prostaglandin E2 inhibits proliferation but not interleukin-2 production by phorbol ester plus calcium ionophore-activated murine T cell clones

We have investigated the effect of prostaglandin E2 (PGE2) on the T lymphocyte activation pathway. 2. At physiologically attainable concentrations (-0.1 micronM), PGE2 effectively inhibited the proliferation of murine antigen0specific "helper" T cell clones stimulated either with specific antigen in the presence of macrophages or with phorbol ester plus calcium ionophore A23187. The inhibition was not reversed by the addition of exogenous Interleukin 2(IL-2) in either case. 3. PGE treatment at the same concentrations did not inhibit IL-2 production by phorbol ester plus calcium ionophore-stimulated T cell clones as assayed by CTLL proliferation. 4. These results suggest that the major target (or targets) of PGE) inhibition directly on T cells lies in the IL-2 signal transduction pathway rather than in the early activation events leading to T cell activation

Phorbol myristate acetate does not impair the inhibitory branch of adenylate cyclase in hamster adipocytes

La activación de alfa2-adrenoreceptores o Ri-adenosinreceptores en células adiposas disminuyó considerablemente la acumulación de AMP cíclico inducido por el agonista beta-adrenérgico isoprenalina. Esta acción casi fue abolida en células de hamster tratadas con toxina de la tosferina. El ester forbol activo, forbol 12-miristrato 13 acetato, fue incapaz de reproducir el efecto de la toxina en este modelo. Los datos indican que la activación de la proteinoquinasa C en este modelo celular no altera la rama inhibitoria de la adenil ciclasa