Adenosine prevents phorbol ester injury in rabbit lungs: role of leukotrienes and TNF.

The objective of this study was to determine whether adenosine (ADO) prevents phorbol myristate acetate- (PMA) induced lung injury by modulating peptidoleukotrienes (LT) and/or tumor necrosis factor (TNF) production. PMA significantly increased pulmonary vascular resistance (PVR, 275 +/- 4 to 447 +/- 30 cmH2O.1-1.min) and microvascular filtration coefficient.(Kf, 0.024 +/- 0.002 to 0.040 +/- 0.006 g.min-1.cmH2O-1) in isolated blood-perfused rabbit lungs. ADO (5 mumol/min) blocked the increases in PVR (257 +/- 9 to 283 +/- 26) and Kf (0.028 +/- 0.005 to 0.018 +/- 0.002). After PMA (30 min), perfusate levels of LTC4 + LTD4 increased by 15.3 +/- 2.1 pg/ml; LTE4 increased by 15.1 +/- 4.1 pg/ml. ADO reduced the increase in LTC4 + LTD4 to 2.7 +/- 6.1 pg/ml, but total LT increased by 31.9 +/- 16.6 pg/ml, implying that ADO enhanced the conversion of LTC4 and LTD4 to LTE4. MK-886 (L663,536), an LT synthesis inhibitor, blocked the increase in total LT (6.1 +/- 13.9 pg/ml) but did not reduce the PMA-induced increase in Kf (0.022 +/- 0.003 to 0.035 +/- 0.005) or PVR (238 +/- 11 to 495 +/- 21). After PMA administration, perfusate TNF levels were not different from the 10-fold increase observed in control experiments and were not reduced by ADO or MK-886. TNF production was independent of perfusate blood components and presumably due to low levels of endotoxin in the perfusate (70-90 ng/ml). These results indicate that ADO does not protect against PMA-induced acute lung injury by altering circulating levels of LT or TNF.     

Multiple effects of phorbol ester on secretory activity in rabbit gastric glands and parietal cells

Acid secretory activity and respiration in rabbit gastric glands are stimulated by cAMP-dependent and -independent agonists. Potentiation between agonists suggests interaction of the activation pathways. Regulation of secretory response by protein kinase C was investigated with 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA elevated basal respiration, pepsin release, and acid secretion but inhibited histamine and carbachol stimulation of acid secretion by gastric glands, as measured by [dimethylamino-14C]aminopyrine accumulation. The inhibition of histamine response was specific for protein kinase C activators, occurred after a 20-min lag, and was not reversed by removal of TPA after 3 min of preincubation. TPA pretreatment inhibited acid secretory responses to cholera toxin and forskolin but enhanced the response to cAMP analogues. Cholera toxin and pertussis toxin simulated ADP-ribosylation of 45 and 41 kDa proteins, respectively, in parietal cell membranes. Therefore, both stimulatory (Gs) and inhibitory (Gi) GTP binding proteins of adenylyl cyclase appear to be present in parietal cells. Pretreatment with pertussis toxin attenuated PGE2 but not TPA inhibition of histamine stimulation of aminopyrine accumulation. Thus, the inhibitory effect of TPA does not appear to be associated with an action on Gi. The results with histamine and carbachol suggest that protein kinase C may regulate both cAMP-dependent and -independent stimulation of parietal cell acid secretion.     

Kinetics of serotonin uptake in isolated rabbit lungs

The single-pass, bolus-injection method was used to study the effect of serotonin (5-HT) concentration on the extraction of 5-HT by isolated perfused rabbit lungs. The extraction pattern suggested that an uptake model, which includes multiple parallel uptake processes, provided a better representation of the data than the simple Michaelis-Menten equation, which has commonly been used to fit the saturable uptake data in previous studies. In particular, the rabbit lung data could be fit with two such parallel pathways. Since the 5-HT uptake could virtually be completely blocked by imipramine, both pathways can be considered to be carrier-mediated processes. The high-affinity pathway was saturable within the range of concentrations studied, with a Km and Vmax of approximately 0.84 microM and 0.21 nmol.s-1.g wet lung wt-1. The Km for the low-affinity pathway was larger than concentrations for which accurate uptake measurements are practical in the perfused organ. Thus, for the low-affinity pathway, only Vmax/Km was identifiable. Vmax/Km values for the high- and low-affinity pathways were approximately 2.87 and 0.35 ml/s, respectively. The results suggest that it will be worthwhile to investigate the behavior of these uptake parameters in response to changes in lung physiology and endothelial function in future studies.     

Synergistic functions of phorbol ester and calcium in serotonin release from human platelets

In human platelets, thrombin activates Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and mobilizes Ca2+ concomitantly, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) may be intercalated into membranes and directly activates protein kinase C without mobilization of Ca2+ in sufficient quantities. A series of experiments with TPA and Ca2+-ionophore (A23187) indicates that activation of protein kinase C is a prerequisite requirement for release of serotonin, and that this enzyme activation and Ca2+ mobilization act synergistically to elicit a full cellular response. Both cyclic AMP and cyclic GMP inhibit activation of protein kinase C by prohibiting the signal-dependent breakdown of inositol phospholipid to produce diacyl-glycerol, but none of these cyclic nucleotides prevents the TPA-induced activation of this enzyme.     

The effect of a phorbol ester on amphibian myogenesis

The effect of a phorbol ester (TPA) on myogenesis of urodelian embryo has been investigated. Presumptive somites were extirpated from mid-neurula, the stage when they have already attained certain degree of determination. The explants were treated with TPA (10 ng/ml in Steinberg solution with the addition of 10% Leibovitz's L-15) for 4 days, the period during which the frequency and size of gap junctions in normal myogenic cells are at the maximum. After the treatment many cells became dispersed and isolated while others spread out and remained connected in the form of a sheet. During further culture for 6 days in the solution without TPA myogenesis was blocked. Both the dispersed cells and the cells in a sheet remained at the earliest stage of muscle differentiation with irregular shape or in the form of myoblast, while cells of the control series formed myotubes containing bundles of myofibrils. The result suggests that the blockage of myogenesis is the consequence of interruption of junctional communication evoked by TPA treatment.     

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.     

Carbonic anhydrase activity of rabbit lungs

Pulmonary carbonic anhydrase (CA) activity was studied in rabbit lungs perfused with solutions containing no CA. Measurements were made of the amount of 14CO2 appearing in the expired gas following injections of H14CO3(-), 14CO2, or a 20:1 mixture of each into the pulmonary artery. The fraction of the injected label in the expired gas was only 17% greater for 14CO2 than for the mixture, suggesting that equilibration between H14CO3(-) and 14CO2 was nearly complete during the capillary transit time. Inhibition of pulmonary CA decreased excretion of H14CO3(-) and the mixture by 40 and 49% and increased the excretion of 14CO2 by 96%. Addition of CA to the perfusate had no effect. Thus, CO2 exchange is not significantly limited by pulmonary CA if inhibitors are absent. Tissue binding of [3H]acetazolamide injected into the pulmonary artery was diminished by 50% when acetazolamide concentrations reached 0.13 x 10(-6) M. Each liter of extravascular lung water contained 1.25 x 10(-6) mol of receptors for acetazolamide that were accessible to plasma during a single circulation. Binding of [3H]acetazolamide was also observed in lungs of anesthetized rabbits, suggesting that pulmonary CA is accessible to plasma in vivo as well as in situ.     

Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester.

Nuclear factor kappa-B (NF-kappa B) has been shown to play an important role in LPS-mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP-dependent protein kinase in the regulation of NF-kappa B activity. In this study we have investigated the mechanism of NF-kappa B induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF-alpha NF-kappa B element was transfected into the RAW264 macrophage-like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8-bromo cAMP. To further study NF-kappa B induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF-kappa B binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA-binding activity confirmed these results and indicated that LPS induced a 55 KD DNA-binding protein. Induction of this NF-kappa B binding activity was not inhibited by pretreatment with the PKC inhibitor H-7. H-7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down-regulates PKC, had no effect on LPS induction of NF-kappa B activity in these cells. These results suggest that the induction of NF-kappa B in macrophages by LPS is independent of PKC.     

Aldosterone secretion: effect of phorbol ester and A23187

The effects of the divalent ionophore, A23187, the phorbol ester, and/or 12-0-tetradecanoyl-phorbol-13-acetate on aldosterone secretion from adrenal glomerulosa cells were compared to those of angiotensin II (AII). AII causes a prompt and sustained increase in secretion. A23187 causes an initial increase followed by a gradual decline to values less than 25 percent of those seen with AII. TPA causes no initial increase but a slowly progressive rise in secretion rate to a less than maximal value. When TPA and A23187 act together, there is a prompt and sustained increase in aldosterone production rate similar to that seen after AII addition. The effect of TPA is dependent on the free Ca2+ concentration of the cell cytosol. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system operate to control respectively the initial and sustained phases of the secretory response. The first phase occurs as a consequence of amplitude modulation of the calmodulin branch of the system by a rise in [Ca2+]c, and the second phase as a consequence of the sensitivity modulation of the C-kinase branch by diacylglycerol.     

Role of calcium in the potentiating effect of phorbol ester on KCl-induced vasocontraction

The mechanism of the potentiating effect of phorbol ester on potassium-induced contraction in rat aorta was investigated. The contractile response to KCl in the medium containing 0.5 mM CaCl2 was significantly increased by pretreatment with 10(-8) M phorbol 12-myristate 13-acetate (PMA), but not with 10(-7) M 4 alpha-phorbol. The dose-response curve to calcium in 30 mM KCl-induced contraction was shifted to the left by PMA pretreatment and the EC50 value (the concentration producing a half maximal response) of calcium was significantly lower in aorta pretreated with PMA than in the control. On the other hand, calcium influx stimulated by 30 mM KCl was not changed by PMA pretreatment. Both the contractile response and the corresponding calcium influx induced by 30 mM KCl were abolished by preincubation with 10(-6) M verapamil for 45 min. These results suggest that activation of protein kinase C potentiates the contractile response to KCl by increasing the sensitivity of the intracellular contractile apparatus for calcium.     

Analysis of phorbol ester receptors in phorbol ester unresponsive 3T3 cell variants

The phorbol ester tumor promoters induce multiple cellular responses in cell culture, including mitogenesis. We have analyzed 3 variants of mouse 3T3 cells mitogenically unresponsive to the phorbol esters for phorbol ester receptors. All resembled control 3T3 cells in their specific [³H]phorbol 12,13-dibutyrate binding. The variants thus appear to be altered at steps distal to receptor occupancy in the mitogenic response to the phorbol esters.     

Role of protein kinase C in regulating smooth muscle electrical and contractile activity: the effect of phorbol ester

The effects of protein kinase C activation by 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) on the functions of guinea-pig smooth muscle taenia coli have been studied, using double-sucrose-gap method. A 15-20-min treatment of the muscle with 2 X 10(-8) M TPA caused a progressing inhibition of spontaneous electrical activity and mechanical tension, suppression of post-hyperpolarizing electrical and contractile "off-responses", a decrease in the number of action potentials during superthreshold membrane depolarization, depression of electrical and mechanical responses induced by acetylcholine, histamine, bradykinin mediators. The treatment of pre-depolarized (140 mM kappa+) muscle with 2.10(-8) TPA has led to a considerable reduction in contractile responses induced by the above mediators. The results obtained indicate that protein kinase C is capable of regulating both voltage-sensitive and receptor-operated ionic channels in smooth muscle cells.     

Differential effect of diacylglycerols and phorbol ester on arachidonic acid metabolism in bone marrow-derived macrophages

Murine bone marrow-derived macrophages were induced to prostaglandin synthesis by activators of protein kinase C, the phorbolester TPA and the diacylglycerols dioctanoylglycerol (diC8) and diolein (diC18:1). As short term stimulation of prostaglandin synthesis is mainly dependent on the availability of free arachidonic acid, the modulation of arachidonic acid liberation and reacylation was investigated. DiC8 inhibited the reacylating enzyme lysophosphatide acyltransferase in the in vitro assay, but there was no evidence for an inhibitory effect of TPA or diacylglycerols on the activity of the lysophosphatide acyltransferase in whole cells. The release of arachidonic acid from prelabelled cells was stimulated by TPA and the diacylglycerols even in the presence of an inhibitor of reacylation, indicating an activation of phospholipase A2. An activation of phospholipase A2 was measured in membranes derived from TPA-stimulated macrophages. These data indicate that the enhanced pool of free arachidonic acid, which drives prostaglandin synthesis, is primarily due to a stimulation of the liberation of arachidonic acid from membrane phospholipids.     

A phorbol ester and a daphnane ester stimulate a calcium-independent kinase activity from human mononuclear cells

TPA and a non-promoting, pro-inflammatory ester RX were used to stimulate the forms of PKC isolated from human mononuclear cells. Three peaks of kinase activity corresponding to gamma, beta and alpha PKC were stimulated by TPA in the presence and absence of calcium and/or phosphatidylserine (PS) but were not activated by RX. A fourth peak eluted at high phosphate concentration was activated by TPA and RX in the presence of PS and the absence of calcium. Activity in this fraction was labile to freezing and thawing and was inhibited by staurosporine.     

Influence of vasoconstriction, temperature, and flow on the kinetics of serotonin uptake in rabbit lungs

In the process of estimating the kinetic parameters of the pulmonary endothelial serotonin (5-HT) uptake, it is critically important to distinguish the effects of hemodynamic changes from endothelial injury. Therefore, the effects of changes in flow rate (1.7-5.0 ml/s), hemodynamics (vasoconstriction by norepinephrine), and temperature (39 vs. 33 degrees C) were investigated in isolated rabbit lungs. Indicator-dilution data were expressed in terms of the Michaelis-Menten equation for the two 5-HT uptake pathways in the preparation. The maximum uptake velocity (Vmax1) and the 5-HT concentration at half-maximum velocity (Km1) of the first pathway as well as the first-order constant (Vmax2/Km2) of the linear part of the second pathway were determined. Neither vasoconstriction nor flow variations had any effect on Km1, whereas increasing the flow rate caused extensive recruitment, with a concomitant increase in Vmax1 and Vmax2/Km2. Furthermore, all the kinetic parameters were significantly decreased at the lower temperature. We conclude that Km1 is independent of organ hemodynamics (vasoconstriction and flow) but susceptible to changes in 5-HT uptake capacity caused by a change in temperature. Vmax1 and Vmax2/Km2 respond to alterations in 5-HT uptake capacity and perfused organ volume. These are prerequisites to apply kinetic modeling as a method for the investigation of pulmonary endothelial function and integrity.     

Effect of flow and surface area on angiotensin-converting enzyme activity in rabbit lungs

Pulmonary angtiotensin-converting enzyme (ACE) is located on the luminal surface of pulmonary microvasculature. Multiple indicator-dilution techniques have been used to measure pulmonary ACE activity in vivo and in isolated lungs. These studies suggest that ACE activity is depressed in several forms of acute lung injury. Depression of ACE activity may reflect impaired substrate delivery to enzyme sites because of flow-related reduction of perfused surface area. To assess the role of altered microvascular flow and surface area in the measurement of ACE activity, we utilized similar techniques to estimate the apparent Km and Vmax of pulmonary ACE in isolated, Krebs-perfused rabbit lungs. Km is an estimate of the affinity of a synthetic ACE substrate, [3H]benzoyl-phenyl-alanyl-alanyl-proline ([3H]BPAP), for ACE and should not be influenced by the rate of substrate delivery to luminal enzyme sites. Conversely, Vmax is an index of the number of ACE sites and should be influenced by perfusion changes that alter the number of perfused sites (recruitment or derecruitment). When isolated lungs were subjected to physiological maneuvers designed to increase or decrease perfused surface area, apparent Vmax increased or decreased respectively. Apparent Km was not altered by these maneuvers. Km and Vmax were independent of changes in perfusion rate when surface area was held constant. Thus these parameters should be useful in evaluating perfusion changes in normal and injured lungs.     

Protein kinase C activity following exposure to magnetic field and phorbol ester

We examined the separate and combined effects of 60 Hz sinusoidal magnetic fields (MFs) and a phorbol ester on protein kinase C (PKC) activity in HL60 cells. No enhancement in PKC activity was observed when a cell culture was exposed to a 1.1 mT (rms) MF alone or to a combination of MF and 2 μM phorbol 12-myristate 13-acetate (PMA) for 1 h. In a second set of experiments, cells were preexposed to a less than optimal concentration of PMA (50 nM) for 45 min, followed by a 15 min exposure to both PMA and MF. The data showed a greater decrease in cytosolic PKC activity and a larger increase in membrane activity than was induced by either 1 h PMA treatment alone or PMA and sham MF exposure. One logical conclusion from these data is that MFs may be acting in a synergistic manner on a pathway that has already been activated. Therefore, we suggest that MFs, rather than producing biological effects by a new pathway or mechanism of interaction, exert their effect(s) by interacting with already functioning reactions or pathways. If correct, the question of an MF's mechanism of interaction refocuses on how weak fields might enhance or depress a molecular reaction in progress, rather than on finding a new transduction pathway. Bioelectromagnetics 19:469–476, 1998. © 1998 Wiley-Liss, Inc.