The relationships between arachidonic acid (AA) metabolism and chloride secretion were investigated in mucosal preparations of rabbit distal colon. Tissues displayed a significant cyclooxygenase activity already in nonstimulated conditions and incubation with exogenous AA and calcium ionophore A23187 produced a predominant prostaglandin F2 alpha (PGF2 alpha) profile [PGF2 alpha greater than PGE2 greater than thromboxane B2 (TxB2) greater than 6-keto-PGF1 alpha] as assessed by HPLC of tissue homogenates, whereas 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) was not detected in AA- or A23187-stimulated tissues. Radioimmunological assays showed that PGE2 synthesis was time dependent, plateaued at 10 min, and proceeded at rates 15-20 times over TxB2 and 6-keto-PGF1 alpha. Among the PGs produced by colonic mucosa, only PGE2 and, to a lower extent, PGF2 alpha were found to stimulate chloride secretion and cAMP synthesis. Pretreatment with 10 microM 5,8,11,14-eicosatetraynoic acid, a cyclo- and lipoxygenase inhibitor, prevented AA-induced chloride secretion and PG and cAMP synthesis with the same strength as the cyclooxygenase inhibitor indomethacin. No effects were found after preincubation with nordihydroguaiaretic acid, a lipoxygenase blocker with moderate cyclooxygenase inhibitory properties, and caffeic acid, a lipoxygenase inhibitor. 5-HETE (5 microM) had no effect on short-circuit currents (Isc) and chloride transport, but it significantly reduced the increase in Isc, chloride secretion, and PGE2 synthesis elicited by AA or A23187. Platelet-activating factor, reported to stimulate rabbit colon Isc through an indomethacin-sensitive pathway, was not detected at concentrations as low as 10(-10) M.

In the present study, we evaluated whether motility of Kaposi's sarcoma (KS) cells induced by platelet-activating factor (PAF) is dependent on the regulation of adherens junctions components. The results obtained indicate that PAF dose and time dependently reduced the endogenous expression of the main components of the adherens junctions: VE-cadherin, alpha-catenin, and beta-catenin. In addition, PAF initiated events that directly or indirectly up-regulated both the tyrosine and serine/threonine phosphorylation pathways, and both types of phosphorylation of beta-catenin were involved in the motility of KS cells. This motility was abrogated by addition of the tyrosine kinase inhibitor genistein, suggesting that this phosphorylation is an important signal responsible for breaking down the adherens junctions and diminishing the ability of neighboring cells to interact. Furthermore, immunofluorescence analysis showed that beta-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of controls to a diffuse pattern with gap formation in PAF-treated KS cells. In conclusion, the data presented here indicate that PAF induces tumor cell motility by altering cell-cell adhesion through beta-catenin phosphorylation.

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ABSTRACT. Cyclosporine A (CsA), a neutral, highly hydrophobic cyclic peptide with 11 amino acids, is currently the most widely used immunosuppressive drug for preventing graft rejection and autoimmune diseases. Despite its efficacy, the use of CsA is limited by severe side effects, mainly nephrotoxicity and arterial hypertension. Single cell microfluorimetry was used to evaluate the role of CsA on Ca2+ signaling pathway in intact cells of the porcine proximal tubule-like cell line LLC-PK1; the assay of the in vitro activity of the plasma membrane Ca2+ pump (PMCA) was carried out through the preparation and isolation of membranes. The addition of CsA to incubation medium at doses ranging from 0.1 to 2 μM did not change the basal level of intracellular calcium ([Ca2+]i), whereas it affected the [Ca2+]i response to thapsigargin (TG), a powerful inhibitor of microsomal Ca2+ pump. In control studies, 5 μM TG produced a biphasic response: [Ca2+]i peaked with a 60-s lag, and it then declined to a plateau of elevated [Ca2+]i, which remains above basal. However, it became evident that CsA strengthened the Ca2+ response to TG because the addition of 5 μM TG to cells exposed to 400 nM CsA did not affect the peak response to TG, but it markedly affected the subsequent sustained phase ([Ca2+]i = 156 ± 4.84 versus 130 ± 3.28 nmol, mean ± SEM, n = 6, P < 0.001). In membrane preparations, 200 nM CsA brought about, in the presence of 10 μM calmodulin (CaM), a significant decrease of plasma membrane Ca2+ pump (PMCA) activity (46.96 ± 0.26 versus 53.48 ± 1.96 nmol · mg of protein−1 · min−1, n = 6, P < 0.02), a value similar to that obtained in the presence of equimolar amounts of cyclosporine H (CsH), a non-immunosuppressive analogue of CsA. These findings suggest that in this cell line CsA affects the Ca2+ export pathway through the reduction of the PMCA activity with consequent amplification and strengthening of [Ca2+]i response after exposure to agents that trigger intracellular Ca2+ release. The increased cell sensitivity during Ca2+ signaling events ensuing from the impairment of this “defense system” may be regarded as one of the basic mechanisms involved in the development of the side effects induced by CsA. E-mail: vincenzo.calderaro@unina2.it