Concentrations Of PMA Research Articles

Characterization of T cell responses in peanut-allergic patients by polychromatic flow cytometry

Rationale Antigen-specific effector T cells from allergic individuals are rare. Others have reported the use of CFSE to identify antigen-specific lymphocytes. We sought to characterize the phenotype of antigen-responsive CD4 T cells in peanut-allergic individuals. Methods Challenge-proven peanut allergy patients were recruited. PBMC were CFSE-labeled at a range of concentrations. Following culture, cells were stimulated with low (5 ng/250ng) or high (50 ng/1ug) concentrations of PMA and calcium ionophore. Cells were stained with ethidium monazide bromide, then fixed, permeabilized and stained for flow cytometry. Results CFSE titration experiments revealed substantial suppression of lymphocyte responses to both antigen and mitogen in a dose-dependent manner. Low PMA/calcium ionophore stimulation was optimum for the detection of cytokine with minimum cell death. The ability to incorporate the live/dead cell discriminator, EMA, significantly improved the measurement of proliferation and intracellular cytokine (particularly IL-4). There was a consistent two-to-four fold increase in percent of IL-4 +CD3 +CD4 + over spontaneous levels in response to peanut antigen. In contrast, non-peanut allergic individuals had no appreciable IL-4. IL-4 percentage was higher to peanut extract than to SEB, suggesting the phenotype is antigen-specific. Unexpectedly, there was a higher percent IL-4 + lymphocytes among the non-proliferating population. For many patients, the majority of IL-4 + CD3 +CD4 + lymphocytes were undivided and CD25 −. In contrast, the percent of ifn-γ + cells in the zproliferating subset was substantially higher than in the nondividing population. Conclusions Evaluation of allergen-specific lymphocytes is sensitive to methodology. under the conditions of this study, there is an important th2 effector population with low proliferative capacity.

Late effects of the Chernobyl radiation accident on T cell-mediated immunity in cleanup workers.

The main goal of this investigation was to evaluate the abnormal T-cell immunity in cleanup workers who took part in the cleanup after the Chernobyl accident in 1986. Peripheral blood mononuclear cells (MNCs) of apparently healthy cleanup workers (n = 134) were used to analyze the phenotype and proliferative response to mitogens in vitro. Evaluation of the MNC phenotype of cleanup workers did not reveal a significant disturbance in the T-cell subpopulation content except for an increase in CD3+CD16+56+ (NKT) cells. Immunophenotyping of phytohemagglutinin (PHA)-activated MNCs demonstrated suppression of CD4+ T-cell propagation and augmentation of CD8+ T-cell propagation in vitro compared to control individuals. DNA synthesis in the MNCs of cleanup workers was markedly inhibited after activation for 3 days with suboptimal concentrations of PHA, pokeweed mitogen and PMA. In contrast to control individuals, the monocytes of cleanup workers were able to stimulate the proliferation of T cells from healthy individuals but inhibited the proliferation of T cells from cleanup workers. This study affords a better understanding of the response of MNCs to stimulation with suboptimal concentrations of PHA and provides an approach to a more accurate analysis of the immunological disorders found after exposure to radiation from Chernobyl-related activities.

Uncoupling of ATP-induced inositol phosphate formation and Ca 2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells

The regulation of the increase in inositol phosphates (IPs) production and intracellular Ca 2+ concentration ([Ca 2+] i) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 30 min attenuated the ATP- and UTP-induced IPs formation and Ca 2+ mobilization. The concentrations of PMA that gave half-maximal (EC 50) inhibition of ATP- and UTP-induced IPs accumulation and an increase in [Ca 2+] i were 5–10 and 4–12 nM, respectively. Prior treatment of TECs with staurosporine (1 μM), a PKC inhibitor, partially inhibited the ability of PMA to attenuate ATP- and UTP-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Furthermore, analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-α, -βI, -βII, -γ, -δ, -ε, -θ, and -ζ. With PMA treatment of the cells for various times, translocation of PKC-α, -βI, -βII, -γ, -δ, -ε, and -θ from the cytosol to the membrane was seen after 5- and 30-min and 2- and 4-h treatment. However, 6-h treatment caused a partial down-regulation of these PKC isozymes. PKC-ζ was not significantly translocated and down-regulated at any of the times tested. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide (PI) hydrolysis and consequently attenuate the [Ca 2+] i increase or inhibit independently both responses to ATP and UTP. The translocation of PKC-α, -βI, -βII, -δ, -ε, -γ, and -θ induced by PMA caused an attenuation of ATP- and UTP-induced IPs accumulation and Ca 2+ mobilization in TECs.

Signalling components underlying platelet aggregation to a Ca 2+ ionophore and a phorbol ester

Although it is well established that G q - and G i -coupled receptors can combine to mediate platelet aggregation, the signalling events underlying the synergy are not fully characterised. This study has used the calcium ionophore, A23187, and phorbol ester, PMA, to investigate this question. We show that aggregation to submaximal but not maximal concentrations of ionophore is partially inhibited by antagonism of the P2Y 12 ADP receptor or PKC blockade. However, a full aggregation response can be restored under these conditions by addition of PMA or ADP. Studies using PI 3-kinase inhibitors demonstrate that this is the second messenger pathway that restores aggregation by the G i -coupled receptor in the presence of PKC blockade. However, under normal circumstances, PI 3-kinase activity is not a major requirement for aggregation to the ionophore. PMA stimulates a weak aggregation which takes several minutes to reach a maximum. Threshold concentrations of PMA and a G i -coupled receptor agonist when added alone show no effect on aggregation, but when combined induce aggregation responses. This study demonstrates that calcium and PKC interact synergistically with a G i -coupled receptor agonist to mediate aggregation, and also with each other.Activation of G i supports aggregation in part through the PI 3-kinase pathway. High concentration of ionophore on their own can induce aggregation independent of PKC and activation of G i . Multiple signalling pathways mediate platelet aggregation and their relative importance depends on experimental conditions.

Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca 2+ Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca 2+ concentration ([Ca 2+] i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca 2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC 50) and maximal inhibition of BK induced an increase in [Ca 2+] i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca 2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-α, βI, βII, δ, ϵ, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K D and B max of the BK receptor for binding (control: K D = 1.7 ± 0.2 nM; B max = 47.3 ± 4.4 fmol mg protein ), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-α, βI, βII, δ, ϵ, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca 2+ mobilisation in VSMCs.

Regulation of HSD17B1 and SRD5A1 in Lymphocytes

We previously reported lymphocyte expression of genes encoding enzymes required for steroid metabolism; however, only 17β-HSD and 5α-reductase showed significant enzyme activity. We now investigate regulation of lymphocyte expression for genes encoding 17β-HSD and 5α-reductase. Cultured human T and B lymphoid cell lines and peripheral blood mononuclear cells were treated with known regulators of steroidogenic gene expression including forskolin, PMA, ionomycin, various steroids, interleukin (IL)-4, and IL-6. Treatment with 10 or 50 μM forskolin resulted in a 20–60% reduction of expression for HSD17B1 (encoding 17β-HSD I) in T and B lymphoid cell lines and peripheral blood mononuclear cells, although such a change was not observed in the expression of SRD5A1 (encoding 5α-reductase I). No significant changes were found when cells were treated for 24 h with various concentrations of PMA or ionomycin. Incubation with 10−9 to 10−7 M androstenedione or estradiol increased expression of HSD17B1, while testosterone decreased the expression of this gene. SRD5A1 expression was increased in the presence of 5α-DHT although no consistent changes were observed when the cells were treated with testosterone. Other steroids, including dexamethasone, progesterone, and 6-hydroxypregnanolone, produced no effects on expression of either HSD17B1 or SRD5A1. Treatment with 0.1–10 ng/ml of IL-4 or IL-6 also did not effect significant changes in gene expression. These data implicate the involvement of the cAMP–protein kinase signal transduction pathway in regulating lymphocyte expression of HSD17B1. Furthermore, it appears that lymphocyte HSD17B1 and SRD5A1 are regulated to some extent by specific steroids.

Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells.

1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+]i peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+]i were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 microM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, betaI, betaII, gamma, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, betaI, betaII, gamma, delta, epsilon and theta from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 microM PMA for either 30 min or 6 h did not significantly change the KD, and Bmax receptor for BK binding (control: KD=1.7+/-0.3 nM; Bmax=50.5+/-4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses to BK. The translocation of pKC-alpha, betaI, betaII, delta, epsilon, gamma, and theta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

Differential effects of phorbol ester on growth and protein kinase C isoenzyme regulation in human hepatoma Hep3B cells.

PMA has both mitogenic and antiproliferative effects on human hepatoma Hep3B cells. In response to low PMA concentration (10 nM), Hep3B cells displayed an increasing proliferation potentiation. At high PMA concentration (1 microM) Hep3B cells exhibited modest cytostatic effects. Determinations of protein kinase C (PKC) activity in PMA-treated cells revealed that alterations in PKC activity are associated with proliferative capacity. The decrease in PKC activity mediated by a high dose of PMA was accompanied by cell growth inhibition. Increases in PKC activity mediated by a low dose of PMA were consistent with proliferation stimulation. Immunoblot analysis showed that there are at least six PKC isoenzymes: alpha, delta, epsilon, mu, zeta and iota/lambda, constitutively expressed in Hep3B cells. Cellular fractionation and immunocytochemical staining results demonstrated that both 10 nM and 1 microM PMA treatments induced a marked translocation of PKC-alpha from cytosol to membrane or nuclear fraction within 5-30 min. At the same time PKC-delta and epsilon were translocated from the membrane to nuclear fraction. In addition, prolonged treatment with 1 microM PMA, but not with 10 nM PMA, selectively mediated the down-regulation of these three PKC isoenzymes. The distinct effects of different concentrations of PMA on cell proliferation and PKC-alpha, delta and epsilon isoenzyme modulation support the involvement of these three PKC isotypes in the mechanism of action of Hep3B cells in cell growth events.

Cytokines and cross-linking of sIgM augment PMA-induced activation of human leukaemic CD5+ B cells.

Purified leukaemic CD5+ B cells obtained from patients with B cell chronic lymphocytic leukaemia (B-CLL) undergo activation and differentiation following in vitro stimulation with optimal concentrations of the phorbol ester PMA. This paper examines the ability of exogenous cytokines, anti-Ig antibodies, or combinations of these, to enhance or replace the activation signal provided by PMA to different populations of leukaemic B cells. Proliferation induced by PMA was enhanced 2-20-fold when the cells were co-cultured with either anti-Ig, IL-2, IL-4, IL-13, IFN-gamma or TNF-alpha. Moreover, the combination of anti-Ig, PMA and any one of the above cytokines further enhanced proliferation. Anti-Ig and exogenous cytokines were also capable of inducing proliferation in leukaemic B cells cultured with a non-mitogenic concentration of PMA. When taken together with the finding that IL-2, IL-4, IL-13, IFN-gamma and TNF-alpha prevent in vitro apoptosis of leukaemic CD5+ B cells, the results presented here suggest that these cytokines, in conjunction with signals delivered via sIg, may play a role in the proliferation of leukaemic B cells in vivo and, consequently, the pathogenesis of B-CLL.

Protein kinase C plays an important role in the human zona pellucida-induced acrosome reaction.

To investigate the involvement of protein kinases in signal transduction in the human zona pellucida (ZP)-induced acrosome reaction (AR), the effects of protein kinase (PK) activators, dibutyryl cAMP (PKA) and cGMP (PKG), phorbol 12-myristate 13-acetate (PMA, PKC), and the PKC inhibitor, staurosporine were studied. Sperm samples were obtained from normozoospermic men with normal sperm-ZP binding. Oocytes were obtained from other patients with failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were pre-incubated with 2.5-20 microM PMA, 1 mM dibutyryl cAMP or cGMP, 3 mM pentoxifylline or 0.125-2.0 microM staurosporine for 30 min and then incubated with four oocytes for 2 h in human tubal fluid supplemented with bovine serum albumin. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small bore pipette and the state of the AR was determined by fluorescein-labelled Pisum sativum agglutinin. Motility and movement characteristics were assessed by computer-assisted sperm analysis (CASA) after incubation of spermatozoa with PMA for 30 min and 2 h. The dibutyryl cAMP and cGMP analogues had a small positive effect (P < 0.05) but pentoxifylline had no effect on stimulating the ZP-induced AR (P > 0.05). In contrast, PMA stimulated ZP-induced AR in a marked dose-dependent manner. Only the highest concentrations (15-20 microM) of PMA significantly decreased percentage motility (P < 0.001). Doses of 2.5-15 microM of PMA significantly stimulated ZP-induced AR without decreasing motility (P < 0.001). The PKC inhibitor, staurosporine (0.125-0.25 microM) significantly inhibited ZP-induced AR without affecting motility (P < 0.001). Sperm samples from 33 normozoospermic men were used for studies of the ZP-induced AR augmented with 15 microM PMA. One sample did not show a response to PMA stimulation. Among the 14 men with low ZP-induced AR, half had normal responses to PMA and other half had low responses to PMA. In conclusion, activation or inhibition of PKC significantly increases or decreases human ZP-induced AR suggesting that PKC plays a important role in the signal transduction pathway for the physiological AR.

Determination of intracellular cytokines by flow-cytometry following whole-blood culture

Various methods have been reported for measuring intracellular cytokines in peripheral blood mononuclear cells isolated by density-gradient centrifugation. In this report, we describe a whole-blood method for the determination of intracellular cytokines (IFN- γ, TNF- α and IL-2) that uses small-volume (500 μl) blood samples. Directly conjugated anti-cytokine antibodies and commercial cell membrane fixation and permeabilisation reagents were used. Blood was cultured in a 1:3 dilution with a combination of PMA and ionomycin to reveal the cytokine synthetic potential of each cell, together with monensin to increase the sensitivity by retaining cytokines within the cell to detectable levels. The optimum concentrations of PMA (10 ng/ml (16.2 nmol/l)), ionomycin (2 μmol/l) and monensin (3 μmol/l) were determined. Kinetic studies showed maximal cytokine expression after 2 h of culture for TNF- α and IFN- γ and 4 h for IL-2. Assessment of TNF- α and IFN- γ production within the CD4 and CD8 lymphocytes from 10 normal volunteers showed that considerably more CD8+ than CD4+ cells produced IFN- γ. This technique could be used by routine immunology laboratories and will be of use in studies to determine whether cytokine assays are of value in the investigation of immune disorders.

Effects of 5-oxo-6,8,11,14-eicosatetraenoic acid on expression of CD11b, actin polymerization, and adherence in human neutrophils.

Human neutrophils contain a highly specific dehydrogenase that converts 5-hydroxy-6,8,11,14-eicosatetraenoic acid to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). 5-Oxo-ETE is a potent stimulator of calcium mobilization, chemotaxis, and aggregation in these cells and has similar effects on eosinophils. The primary objectives of the current study were to determine whether this compound could increase the surface expression of integrins and stimulate actin polymerization in neutrophils. 5-Oxo-ETE stimulated the expression of CD11b and, to a lesser extent, CD11c, on neutrophils, but had no significant effects on the expression of CD11a, CD16 (Fc gammaRIII), or CD32 (Fc gammaRII). Surface expression of CD11b in response to 5-oxo-ETE was maximal after 12 min and remained constant thereafter. The EC50 for this response (50 nM) was lowered to 20 nM by preincubation of neutrophils with PMA. 5-Oxo-ETE (EC50, 10 nM) also rapidly stimulated actin polymerization in neutrophils, with a maximal response at 20 s. This response was blocked by pretreatment of neutrophils with the Gi protein inhibitor, pertussis toxin, and by homologous desensitization due to preincubation with 5-oxo-ETE. However, preincubation with leukotriene B4 or platelet-activating factor had no effect on the response of neutrophils to subsequent addition of 5-oxo-ETE. The adherence of neutrophils to plasma-coated plastic was also stimulated by 5-oxo-ETE with a time course similar to that for the surface expression of CD11b. Low concentrations of PMA (0.3 nM) enhanced this response. These results raise the possibility that 5-oxo-ETE could contribute to the infiltration of neutrophils into inflammatory sites.

The effect of 12-myristate 13-acetate phorbol ester on human sperm hyperactivation

Objective: To determine whether 12-myristate 13-acetate phorbol ester (PMA) can increase hyperactivated motility of human sperm. Design: A controlled pharmacological study using computer-assisted semen analysis. Setting: Andrology laboratory in a medical research institution. Patient(s): Normal semen was obtained from 48 men. Intervention(s): Washed sperm were exposed to different concentrations of PMA alone or with P and pentoxifylline (PTX) for up to 2 hours and sperm motility measured by a computerassisted semen analyzer. Main Outcome Measure(s): The percentage of sperm with hyperactivated motility was determined from the motility parameters: curvilinear velocity, linearity, and maximum amplitude of lateral head displacement. Result(s): Phorbol ester PMA increased hyperactivated motility in a dose- and time-dependent manner. At 1 hour, the average increases in hyperactivated motility were as follows: 2 μM, 4.8% ± 1.5%; 6 μM, 9.6% ± 1.5%; and 20 μM, 11.3% ± 2.2%. The PMA effect was not altered when P or PTX were added although each separately had a positive effect on hyperactivation. Conclusion(s): Phorbol ester PMA stimulates human sperm hyperactivated motility, indicating the involvement of protein kinase C in the signal transduction pathway.

Secretagogues Increase the Expression of Surfactant Protein A Receptors on Lung Type II Cells

Since secretagogues have been shown to increase the internalization of surfactant phospholipid and protein by lung cells, we postulated that their action occurred through a mechanism involving increased surfactant protein A (SP-A) receptor density. Therefore, we evaluated the influence of secretagogues on the binding of iodinated SP-A to alveolar type II cells. Type II cells were isolated from rat lung and maintained in primary culture for 18 h on Transwell membranes. Upon exposure to 8-bromo-cyclic AMP (cAMP, 0.1 mM), phorbol 12-myristate 13-acetate (PMA, 10 nM), terbutaline (0.1 mM), or ATP (1 mM), the binding of SP-A increased 1.5-2-fold. This stimulation was cell substrate-dependent since type II cells plated on plastic dishes did not show this effect. A time course of the stimulation of SP-A binding due to secretagogues showed that both cAMP and PMA increased SP-A binding by 2-fold after 20 min. With cAMP, binding remained elevated for 2 h, while binding in the presence of PMA had returned to control values. The effects of submaximal concentrations of cAMP and PMA on binding were additive. Inhibition of cellular protein synthesis with cycloheximide did not alter the increase of SP-A binding stimulated by the secretagogues. Type II cells pretreated with PMA responded to subsequent treatment with cAMP by increasing SP-A binding, while these cells were refractory to subsequent treatment with PMA. Both constitutive and regulated binding of SP-A to type II cells were sensitive to trypsin. The binding of SP-A to type II cells showed saturation at a concentration of 1 microg/ml SP-A under control and secretagogue-stimulated conditions, with both total and calcium-dependent binding showing a 2-fold increase upon secretagogue exposure. The data are consistent with the hypothesis that secretagogues stimulate surfactant uptake, at least in part, through recruitment of SP-A receptors to the type II cell surface, resulting in an increase in the number of SP-A binding sites.

Mifepristone modulation of ACTH and CRH regulation of bovine adrenocorticosteroidogenesis in vitro

Mifepristone (RU486), bovine corticotropin-releasing hormone (CRH), arginine vasopressin (VP), adrenocorticotropin (ACTH 1–24) and protein kinase activators (forskolin, [FSK]; phorbol 12-myristate 13-acetate, [PMA]) were used in vitro to investigate their direct effect on adrenocorticosteroidogenesis. Bovine adrenocortical fasciculata/reticularis cells (2 x 10 5 viable cells/well) were cultured for 3 d in medium supplemented with 10% fetal calf serum. After incubation for an additional 24 hr in serum-free medium, cells were treated with serum-free medium alone (Control) or various concentrations of ACTH, CRH, VP, FSK, PMA, RU486, and/or various combinations for 1, 2, 4, or 24 hr. Medium content of cortisol and progesterone were determined by radioimmunoassays. ACTH, CRH, FSK, and PMA each stimulated (P < 0.05) secretion of cortisol in time- and dose-related manners. Although these agents stimulated (P < 0.05) secretion of progesterone in a dose-related manner, medium content of progesterone declined (P < 0.05) over time. The minimal effective doses of ACTH and CRH required to stimulate (P < 0.05) secretion of cortisol relative to the Control over a 4-hr culture period were 0.01 nM and 3 nM, respectively. Relative to observations at 1 hr posttreatment, 24-hr treatment with ACTH or CRH increased the medium content of cortisol by an additional 19.8- and 48-fold, respectively (whereas content of progesterone declined over that time period). VP-stimulated secretion of cortisol was time- (P < 0.05) but not dose-related. Specifically, by 24-hr posttreatment, the medium content of cortisol was increased (P < 0.05) 4.6-fold relative to the quantity of cortisol secreted by 1-hr postaddition of VP (0.01 to 1 μM). Co-treatment with RU486 (1 μM) decreased (P < 0.05) FSK-, ACTH- and CRH-stimulated secretion of cortisol by 77, 27, and 56%, respectively. Similarly, the stimulatory effects of ACTH and CRH on progesterone secretion were reduced (P < 0.05) by 40 and 22%, respectively, by co-addition of RU486. The inhibitory action of RU486 on production of cortisol was no longer apparent by 24 hr after treatment. These observations indicate that RU486 can act as a steroid agonist and as well as an antagonist. These data characterize time- and dose-related direct actions of ACTH, CRH, and RU486 on adrenocorticosteroidogenesis. This information will assist efforts to clarify complex intra-adrenal interactions of neurohormones, growth factors, and endogenous steroids.

Inhibitory Effect of Phorbol Ester on Carbachol-Induced Signal Transduction in Cultured Canine Tracheal Smooth Muscle Cells.

Regulation of the increases in inositol 1,4,5-trisphosphate (IP(3)) production and intracellular Ca(2+) concentration ([Ca(2+)](i)) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP(3) formation and caused an initial transient peak of [Ca(2+)](i) followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 &mgr;M) for 30 min blocked the carbachol-induced IP(3) formation and Ca(2+) mobilization. Following preincubation, carbachol-induced Ca(2+) mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP(3) formation and increase in [Ca(2+)](i) were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 &mgr;M), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 &mgr;M, did not inhibit these responses to carbachol. The K(d) and B(max) of the muscarinic receptor for [(3)H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca(2+)](i) increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity. Copyright 1995 S. Karger AG, Basel

Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization induced by endothelin-1 in cultured canine tracheal smooth muscle cells

Regulation of the increase in inositol 1,4,5-trisphosphate (IP 3) production and intracellular Ca 2+ concentration ([Ca 2+] i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by endothelin-1 (ET-1) led to IP 3 formation and caused an initial transient peak followed by a sustained elevation of [Ca 2+] i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 μM ) for 30 min blocked the ET-1-induced IP 3 formation and Ca 2+ mobilization. However, this inhibition was reduced after incubating the cells for 8 h with PMA. Following preincubation, ET-1-induced Ca 2+ mobilization recovered with time and reached the same extent of control cells within 48 h. The concentrations of PMA that gave half-maximal inhibition (-IogEC 50) of ET-1-induced IP 3 formation and increase in [Ca 2+] i were 8.6 and 8.4 M, respectively. Prior treatment of TSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate ET-1-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the ET-1-induced IN formation and Ca 2+ mobilization, a change of PKC activity was observed in TSMCs. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca 2+] i increase or inhibit independently both responses. The PMA-induced inhibition of responses to ET-1 was associated with an increase in membranous PKC activity.

Abrogation of the Suppressive Effects of Dexamethasone by PKC Activation or CD28 Triggering

The suppressive effect of the glucocorticoid dexamethasone (DEX) on purified CD+ T cells was found to depend on the activation pathway. In contrast to anti-CD3- or PHA-induced T cell proliferation, the alternative pathway of T cell activation, i.e., through anti-CD2 and anti-CD28, appeared largely resistant to DEX. By titrating anti-CD28 or the protein kinase C (PKC) activator PMA in the DEX-sensitive systems, it was demonstrated that inhibition by DEX could be abrogated by enhancing the CD28 signal or by stimulation of the PKC-dependent pathway. Supraoptimal concentrations of PMA were inhibitory for proliferation and this effect was partly prevented by DEX. These data suggest that the outcome of the effect of DEX on CD4+ T cells is dependent on the activation pathway, in particular the role and composition of the transcription factor AP-1.

5‐Hydroxytryptamine receptor‐mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor. 7. Acetylcholine-induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5-HT-induced IPs accumulation is not due to release of acetylcholine.8. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis via a pertussis toxin- and cholera toxin-insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5-HT2 receptors may be predominantly mediating IPs accumulation and presumably IP-induced Ca2+ release may function as the transducing mechanism for 5-HT stimulated contraction of tracheal smooth muscle.

The absence of constitutive and induced expression of critical cell-adhesion molecules on human cardiac myocytes. Its role in transplant rejection.

Fetal human cardiac myocytes (FHCM) and a cell line derived from FHCM, termed W1, constitutively express low levels of MHC class I antigens and significant levels of ICAM-1 (CD54), and LFA-3 (CD58) but do not express LFA-1 alpha (CD11a), LFA-1 beta (CD18), GMP-140 (CD62), BB1-B7, VCAM-1, and ELAM-1. In vitro incubation of FHCM or the W-1 cell line for varying periods with varying concentrations of IFN-gamma, TNF-alpha, Poly IC, LPS, IL-alpha, IL-1 beta, PMA, PDBu, and supernatant fluids from Con A-activated PBMC or allogeneic MLR cultures failed to induce cell adhesion molecules (CAMs) or costimulatory molecules that are not constitutively expressed on these cells except for MHC class II antigens. In addition, IFN-gamma, Con A, and MLR supernatant fluids (in order of biological activity) not only induced MHC class II antigens but also markedly increased the mean density of expression per cell of MHC class I and ICAM-1. Analysis of the stability of MHC class I/II molecules using agents like brefeldin-A and Western blot analysis of MHC class II molecules suggest that these ligands are very stably expressed on myocytes. Our previous studies have documented the failure of MHC-expressing FHCM to induce an alloproliferative response. The results of the present studies show that this failure is not secondary to the absence of ICAM-1 or LFA-3 or the presence of unstable MHC molecules but is most likely due to the absence of other CAMs/costimulatory molecules that are critically required for inducing allogeneic activation.