Effects of wortmannin and rapamycin on CSF-1-mediated responses in macrophages.

There are differing views regarding the roles of phosphatidylinositol 3-kinases (PI3-kinases) and p70 S6 kinase (p70s6k) in growth factor-induced cellular responses. One approach that is widely employed to investigate these roles is to use the inhibitors, wortmannin and rapamycin, respectively. This approach is used here to study the responses in macrophages to colony stimulating factor-1 (CSF-1). Wortmannin (> or = 30 nM) and rapamycin (> or = 3 nM) both weakly inhibited CSF-1-stimulated DNA synthesis in murine bone marrow-derived macrophages (BMM), suggesting that there are PI3-kinase- and p70s6k-independent pathways required for the onset of S phase; interestingly the combination of the drugs gave dramatic suppression. Inhibition of DNA synthesis by rapamycin on the BMM was much less than that observed with the CSF-1-dependent cell line, BAC1.2F5. In BMM, wortmannin suppressed CSF-1-stimulated increase in p70s6k activity indicating that PI3-kinase activity may lie upstream. In contrast to some other growth factor/cell systems, no evidence was obtained using the inhibitors for the involvement of PI3-kinase or p70s6k in CSF-1-mediated induction of c-fos mRNA expression or Erk-1 activity; in addition, no evidence was found for an involvement in the CSF-1-mediated increase in cyclin D1 expression or STAT activation. The findings reinforce the need to study the signal transduction cascades relevant to each individual growth factor and preferably not in cell lines.

Inositol phosphate turnover in human airways: effect of arachidonic acid metabolism.

The aim of this study was to examine the role of the phospholipase A2-arachidonic acid (PLA2-AA) pathway in response to histamine and the possible effect of AA metabolites on the generation of inositol-phosphates (IPs) in airway smooth muscle cells (SMC). Radiolabelled IPs-were analyzed by high-performance liquid chromatography (HPLC). Indomethacin, a cyclooxygenase inhibitor, potentiated the IP production evoked by a 5-sec application of histamine, while it decreased the effect of a 5-min incubation with the agonist, suggesting a time-dependent modulation of phospholipase C (PLC) activity by prostaglandins (PGs). Exogenous AA elicited the generation of IPs; this effect was suppressed by cyclooxygenase inhibition. Therefore, several steps in the AA metabolic pathways appear to modulate the production of IPs in human airway SMC.

Control of inositol phosphate turnover in human airways during histamine stimulation.

The control of inositol phosphate (IP) turnover was investigated in intact human airway smooth muscle cells (SMC) during a brief exposure to a bronchoconstrictor agonist. The pool of membrane phosphatidylinositol 4,5-biphosphate was labelled by incubating SMC with myo-[3H]inositol and the [3H]IPs synthetized ([3H]1,4-IP2, [3H]1,3,4,-IP3, [3H]1,4,5,-IP3 and [3H]1,3,4,5-IP4) were separated by HPLC. We examined the role of protein kinase C (PKC) and of Ca2+ on IP turnover during a 5 sec application of histamine. Activation of PKC with the phorbol ester PMA (0.2 microM) decreased, whereas inhibition of PKC with 1 microM staurosporine increased the production of the 4 IPs examined in unstimulated and in histamine-stimulated SMC. Decreasing [Ca2+]i with 5 microM ionomycin in the absence of external Ca2+ diminished the IP production whereas in the presence of Ca2+, ionomycin exalted it and potentiated the response to histamine. Thapsigargin, 5 microM, which depletes the 1,4,5-IP3-sensitive Ca2+ stores, reduced the IP production due to histamine. The effects of PMA, staurosporine and thapsigargin were also tested on [Ca2+]i in fura-2-loaded single SMC. These results reveal that PKC exerts a negative and Ca2+ a positive feedback control on phospholipase C, that operate within 5 sec of agonist stimulation.

Arachidonic acid induces [Ca2+]i oscillations in smooth muscle cells from human airways.

The role of phospholipase A2 (PLA2) in mediating agonist-stimulation of bronchial contraction has not yet been studied at a cellular level. The aim of the present work was to examine the Ca2+ signal elicited in human bronchial smooth muscle cells by exogenous arachidonic acid (AA), a first product of PLA2 activity. To study the effect of AA (2-20 microM) on [Ca2+]i, a single cell video imaging technique was used. AA induced various patterns of [Ca2+]i increase; most frequently, [Ca2+]i oscillations were observed. The amplitude of [Ca2+]i repetitive peaks, but not the frequency, was dependent on AA concentration. In the absence of external Ca2+, AA evoked a slower and reduced transient increase in [Ca2+]i. It is concluded that AA or some of its metabolites release Ca2+ from intracellular stores and that a contribution of extracellular Ca2+ is essential for [Ca2+]i oscillations.

Caldesmon, calponin and alpha-smooth muscle actin expression in subcultured smooth muscle cells from human airways.

Calponin and caldesmon are two proteins considered to play a regulatory role in smooth muscle contraction, which have never previously been found to be expressed in subcultured cells. In the present study, immunocytochemistry and immunoblotting were performed to identify these proteins in smooth muscle cells (SMC) from human bronchi. It was found that human airway SMC, kept in a non-proliferative state, continued to express caldesmon and calponin at least until the 8th passage. The expression of alpha-smooth muscle actin studied under the same conditions was also shown to be preserved in subcultured bronchial SMC.

Signal transduction in smooth muscle cells from human airways.

The aim of this study deals with the post-receptor events involved in the response of cultured smooth muscle (SMC) from human bronchi to various agonists of the contraction. [3H]inositol phosphates (IPs) were isolated by high performance liquid chromatography (HPLC) and intracellular Ca2+ concentration ([Ca2+]i) was determined with the Fura-2 fluorescence technique. Following 5 sec stimulation with histamine, an elevation of several [3H]IPs, in particular [3H]1,4-IP2, [3H]1,4,5-IP3 and [3H]1,3,4,5-IP4, above the control values was observed. Following an incubation of 10 or 15 sec with histamine, the content of [3H]1,4,5-IP3 declined towards its basal value, while the amount of metabolites ([3H]4-IP, [3H]1,4-IP2, [3H]1,3,4-IP3) increased with time; [3H]1,3,4,5-IP4 varied little between 5 and 10 sec and decreased at 15 sec. SMC responded also to carbachol and to prostaglandin F2 alpha (PGF2 alpha) by an enhanced production of [3H]IPs, whereas neurokinin A (NKA) had no effect on the turnover of [3H]IPs. Histamine, carbachol and PGF2 alpha evoked a transient elevation in [Ca2+]i, followed by a sustained increase. The duration of the transient elevation appeared similar to that of the increase in [3H]1,4,5-IP3. These results suggest that the 'phospholipase C-1,4,5-IP3-Ca2+ release' signalling pathway is involved in the physiological response of human airway SMC to histamine, carbachol and PGF2 alpha.

Agonist-induced production of inositol phosphates in human airway smooth muscle cells in culture.

Smooth muscle cells (SMC) from human bronchi were isolated by elastase treatment, subcultured, and characterized by their positive reaction with a monoclonal antibody against alpha-smooth muscle actin (alpha SMA). In each cell line tested, at least 95% of the cells were positively stained. The functional properties of these cells were examined by measuring the metabolism of inositol phosphates (IPs). For that purpose, cells were incubated for 3 days before reaching confluency in the presence of myo-[3H]inositol in order to label the phosphoinositide pool, and the various [3H]IPs were separated by HPLC on a SAX column with a phosphate gradient. IP1 isomers were separated in three peaks; IP2, IP3, IP4, IP5 and IP6 (phytic acid) were each eluted as single peaks. The identity of the [3H]peaks was verified with corresponding [3H]IP standards. The accumulation of [3H]IPs was measured by incubating cells up to 30 min in the presence of 10 mM LiCl, with or without a bronchoconstrictor agent (carbachol, histamine, PGF2 alpha). Histamine, 10(-4) M, elicited a four times larger IP accumulation than carbachol, 10(-4) M, and than PGF2 alpha, 5 10(-5) M. Dose-response curves were established for histamine and carbachol in the range 10(-7)-10(-4) M. At 10(-7) M, carbachol was more effective than histamine in stimulating the IP metabolism. Atropine blocked the response to carbachol, and diphenhydramine inhibited the effect of histamine, indicating the specificity of the response to the agonists. These results indicate that cultured human bronchial SMC are a suitable preparation for studying physiological aspects of membrane transduction in the airways.

Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes.

We have investigated the possible role of plasma membrane oxidoreductases in the Ca2+ export mechanisms in rat brain synaptic membranes. Ca2+ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca2+ pump and by a low affinity/high capacity ATP-independent Na(+)-Ca2+ exchanger. Both Ca2+ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP greater than NAD greater than NADPH greater than NADH with IC50 values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca2+ pump and with IC50 values between 8 and 10 mM for the Na(+)-Ca2+ exchanger. Oxidizing agents such as DCIP3 and ferricyanide inhibited the ATP-driven Ca2+ efflux mechanism but not the Na(+)-Ca2+ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca2+ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na(+)-Ca2+ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by either cAMP-dependent protein kinase, Ca2+ calmodulin-dependent protein kinases, or protein kinase C.