Regulation of immunoglobulin E-mediated secretion by protein phosphatases in human basophils and mast cells of skin and lung.

A wide range of serine/threonine protein phosphatase (PP) inhibitors were studied for effects on the immunoglobulin E (IgE)-mediated release of histamine from human lung mast cells, human skin mast cells and basophils. Okadaic acid (OA) inhibited the release of histamine from all three cell types in a concentration-dependent manner. Two structural analogues of okadaic acid, okadaol and okadaone, known to be less active than the parent molecule as inhibitors of PP, were less active than okadaic acid as inhibitors of histamine release in these three cell types. A number of PP inhibitors, showing differences in selectivity for PP1 and PP2A, were also evaluated. Calyculin, which is roughly equipotent as a PP1 and PP2A inhibitor, attenuated the release of histamine from all three cell types. Similarly, tautomycin (TAU), which shows greater selectivity for PP1 over PP2A, was also effective at inhibiting histamine release in all three cell types. In contrast, fostriecin, which is very much more potent as an inhibitor of PP2A over PP1, was ineffective as an inhibitor in all three cell types. These data indicate that the regulation of mediator release by PPs is similar in lung mast cells, skin mast cells and basophils. Moreover, the data suggest that PP1 is important in the control of cellular activity.     

Characterization of protein serine/threonine phosphatase activities in human lung mast cells and basophils

1. The serine/threonine protein phosphatase (PP) inhibitors, okadaic acid and calyculin, attenuated the IgE-mediated release of histamine from human lung mast cells (HLMC) and basophils in a dose-dependent manner whereas an alternative PP inhibitor, microcystin, was ineffective. Calyculin was more potent than okadaic acid in both cell types. The concentration required to inhibit by 50% (IC₅₀) the release of histamine was 15 (HLMC) and 50 nM (basophils) for calyculin and 200 (HLMC) and 300 nM (basophils) for okadaic acid.
2. Lysates of purified HLMC and basophils dephosphorylated radiolabelled glycogen phosphorylase, a substrate for both PP1 and PP2A. The PP activity in lysates of both cell types was inhibited in a dose-dependent fashion by the PP inhibitors with the following rank order of activity, calyculin (approximate IC₅₀; 0.02–0.1 nM)⩾microcystin (0.1 nM)>okadaic acid (70 nM).
3. The PP1-selective inhibitor, inhibitor-2 (I-2), attenuated the dephosphorylation of glycogen phosphorylase in lysates of both HLMC and basophils. I-2 (20 nM) inhibited the glycogen phosphorylase PP activity by 71±3% and 49±13% in HLMC and basophil extracts, respectively. There were, approximately, 6 fold greater levels of I-2-sensitive activity in HLMC than in basophils. Qualitatively similar results were obtained with an alternative PP1-selective inhibitor, inhibitor-1 (I-1).
4. Lysates derived from HLMC and basophils dephosphorylated radiolabelled casein which is a PP2A-restricted substrate. HLMC lysates contained, approximately, 2.5 fold higher levels of casein PP activity than basophil lysates.
5. These data indicate that HLMC and basophils both contain PP1 and PP2A. The data suggest that, on a per cell basis, HLMC have higher levels of both PP1 and PP2A. Moreover, the ratio of PP1 to PP2A is higher in HLMC than in basophils.

     

Effect of okadaic acid on histamine release from rat peritoneal mast cells activated by anti-IgE

Abstract— The effect of okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A, on histamine release from mast cells has been investigated. Okadaic acid strongly and dose-dependently inhibited histamine release from mast cells induced by anti-IgE. The IC50 value of okadaic acid on histamine release induced by anti-IgE was 3·2 Nm. However, okadaic acid failed to inhibit histamine release induced by A23187 and compound 48/80. Moreover, okadaic acid showed no effect on the initial rise in intracellular Ca²⁺, Ca²⁺-mobilization from intracellular Ca²⁺-stores and the generation of inositol trisphosphate. These results suggest a possible involvement of protein phosphatase 2A in the histamine release from mast cells induced by anti-IgE.     

Inhibitory effect of a toxin okadaic acid, isolated from the black sponge on smooth muscle and platelets.

1. Effects of okadaic acid, a toxin isolated from marine sponges, on smooth muscle contraction and platelet activation were examined. 2. Contractions in rabbit aorta induced by high concentrations of K+ and noradrenaline were inhibited by 0.1-1 microM okadaic acid in a concentration-dependent manner. Spontaneous rhythmic contractions as well as high K+-induced contraction in guinea-pig taenia caeci were also inhibited by 1 microM okadaic acid. 3. High K+-induced contraction in rabbit aorta was accompanied by increased Ca2+ influx measured with 45Ca2+ and increased cytosolic Ca2+ [( Ca2+]cyt) measured with fura-2-Ca2+ fluorescence. Okadaic acid inhibited the contraction without inhibiting Ca2+ influx and produced only a small decrease in [Ca2+]cyt. 4. In a saponin-skinned taenia, Ca2+-induced contraction was not inhibited but rather potentiated by okadaic acid. 5. Okadaic acid, 1 microM, inhibited aggregation, ATP release and increased in [Ca2+]cyt induced by thrombin in washed rabbit platelets. Okadaic acid itself did not change the platelet activities. 6. Okadaic acid did not change the cyclic AMP content of rabbit aorta although the inhibitory effects of okadaic acid were similar to those of cyclic AMP. 7. Although the mechanism of the inhibitory effect of okadaic acid was not clarified in the present experiments, it is suggested that okadaic acid acts by inhibiting protein phosphatases resulting in an indirect activation of cyclic AMP-dependent protein phosphorylation.     

Inhibition of histamine release from human lung and rat peritoneal mast cells by cyclosporin-A.

Cyclosporin A (CS-A) partly inhibited IgE-mediated histamine release from human lung tissue in vitro (chopped and collagenase-dispersed preparations). Inhibition started at concentrations within the clinical blood level of the drug, but the IC50 was much higher (10-50 microM; 50% inhibition reached only in some experiments). CS-A also inhibited histamine release from rat peritoneal mast cells (RPMC) induced by antigen, concanavalin-A (Con-A), compound 48/80 and ionophore A23187. The IC50 values were 0.3, 23.0, and 33.0 microM for Con-A, A23187 and ovalbumin respectively. Inhibition of 48/80-induced release did not reach 50%. By comparison with human basophils the human lung and RPMC were less sensitive to the inhibitory action of CS-A. The IgE-mediated Schultz-Dale reaction in human lung strips was slightly and inconsistently inhibited by CS-A, but IgG1-mediated reaction in guinea-pig lung strips was potentiated by the drug.     

Intradermal actions of hypertonic saline involve neural and vascular mechanisms.

The aim of this study was to investigate whether the wheal and flare responses to intradermal injection of hypertonic (4.5%) saline (HTS) were inhibited by local injection of 1% lignocaine. Eight normal subjects were studied on one occasion. Lignocaine (0.125 ml) was infiltrated at four sites on one forearm and normal saline on the other. Five minutes later, duplicate intradermal injections of 30 microliters of histamine (22.5 nmol ml-1), substance P (1 nmol ml-1), HTS and normal saline were given coded and in random order, one of each pair to each forearm. Lignocaine inhibited flare responses to histamine, substance P and HTS by 56% (P < 0.01), 78% (P < 0.01) and 77% (P < 0.05) respectively suggesting similar involvement of an axon reflex. Wheal to histamine was inhibited by 31% (P < 0.02) and to substance P by 33% (P < 0.05) but not to HTS. This suggests that the mechanism of wheal response to HTS differs from that of histamine and substance P.     

Mechanism of 5-hydroxytryptamine-induced coronary vasodilation assessed by direct detection of nitric oxide production in guinea-pig isolated heart.

1. We assessed whether a submaximal concentration (1 microM) of 5-hydroxytryptamine (5-HT) releases nitric oxide (NO) from the coronary endothelium in guinea-pig perfused heart (n = 5 or 6/group) by direct detection of NO in coronary effluent, and determined whether this accounts for the associated coronary dilation. We also tested whether saponin is a selective and specific tool for examining the role of this mechanism in mediating agonist-induced coronary dilatation. 2. Continuous 5 min perfusion with 5-HT, or acetylcholine (ACh; 1 microM), substance P (1 nM) or sodium nitroprusside (SNP; 1 microM) increased coronary flow from baseline by 3.6 +/- 0.2, 3.4 +/- 0.2, 1.8 +/- 0.1 and 4.1 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Coronary effluent NO content, detected by chemiluminescence, was correspondingly increased from baseline by 715 +/- 85, 920 +/- 136, 1019 +/- 58 and 2333 +/- 114 pmol min-1 g-1, respectively (all P < 0.05). 3. Continuous perfusion for 30 min with NG-nitro-L-arginine methyl ester (L-NAME) 100 microM reduced basal coronary effluent NO content by 370 +/- 32 pmol min-1 g-1 and coronary flow by 7.5 +/- 0.5 ml min-1 g-1 (both P < 0.05). Saponin (three cycles of 2 min of 30 micrograms ml-1 saponin perfusion interrupted by 2 min control perfusion) reduced basal coronary NO content by a similar amount (307 +/- 22 pmol min-1 g-1) but reduced basal coronary flow by only 0.6 +/- 0.2 ml min-1 g-1 (P < 0.05 versus the effect of L-NAME). 4. The increases in coronary flow in response to (5-HT), ACh and substance P were reduced (all P < 0.05) by 100 microM L-NAME to 1.2 +/- 0.3, 1.2 +/- 0.4 and 0.3 +/- 0.3 ml min-1 g-1, respectively. However, the flow increase in response to SNP was not reduced; it was in fact increased slightly to 4.8 +/- 0.4 ml min-1 g-1 (P < 0.05). 5. Similarly, after treatment with saponin, the increases in coronary flow in response to 5-HT, ACh and substance P were reduced to 2.1 +/- 0.3, 1.3 +/- 0.3 and 0.4 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Again, the response to SNP was increased slightly to 4.6 +/- 0.5 ml min-1 g-1 (P < 0.05). 6. L-NAME and saponin also inhibited 5-HT, ACh and substance P-induced NO release (P < 0.05), without affecting equivalent responses to SNP. 7. For substance P, the change in coronary flow (delta CF) correlated with log10 delta NO in the presence and absence of saponin and L-NAME; delta CF = 1.2(log delta NO) 1.9; r = 0.92; P < 0.05. For 5-HT the relationship was delta CF = 2.2(log delta NO-2.7; r = 0.79; P < 0.05, indicating that 5-HT causes a disproportionately greater increase in coronary flow per release of NO. This was taken to indicate that 5-HT relaxes coronary vasculature in part by releasing NO, but in part by additional mechanisms. ACh resembled 5-HT in this respect. 8. Saponin had no effect on cardiac systolic or diastolic contractile function assessed by the construction of Starling curves with an isochoric intraventricular balloon. 9. In conclusion, despite its minimal effect on basal coronary flow, saponin is an effective tool for revealing endothelium-dependent actions of coronary vasodilator substances and has selectivity in that it does not impair endothelium-independent vasodilatation or cardiac contractile function. 5-HT dilates guinea-pig coronary arteries largely by the release of NO from the coronary endothelium.     

Selective antagonism by PPADS at P2X-purinoceptors in rabbit isolated blood vessels.

1. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a P2-purinoceptor antagonist, was investigated for its ability to antagonize: (1) P2X-purinoceptor-mediated contractions of the rabbit central ear artery and saphenous artery evoked by either alpha,beta-methylene ATP (alpha,beta-MeATP) or electrical field stimulation (EFS); (2) P2Y-purinoceptor-mediated relaxations of the rabbit mesenteric artery; (3) endothelium-dependent and endothelium-independent, P2Y-purinoceptor-mediated relaxations of the rabbit aorta. 2. alpha,beta-MeATP (0.1-100 microM) caused concentration-dependent contractions of the rabbit ear and saphenous arteries. The negative log[alpha,beta-MeATP] that produced a contraction equivalent to the EC25 for noradrenaline (ear artery) or histamine (saphenous artery) in the absence of PPADS was 6.60 +/- 0.18 (9) and 6.18 +/- 0.17 (9) in the ear artery and saphenous artery, respectively. These effects of exogenous alpha,beta-MeATP were concentration-dependently inhibited by PPADS (1-30 microM). In the ear artery, the negative log[alpha,beta-MeATP] producing a contractile response equivalent to the EC25 of noradrenaline, in the presence of PPADS at 1, 3 and 10 microM was 6.16 +/- 0.18 (8), 5.90 +/- 0.18 (8) and 4.72 +/- 0.36 (8), respectively (P < 0.01). In the saphenous artery, the negative log[alpha,beta-MeATP] values equivalent to the EC25 for histamine in the presence of PPADS at concentrations of 1, 3, 10 and 30 microM were 5.90 +/- 0.19 (8), 5.73 +/- 0.16 (8), 4.99 +/- 0.14 (8) and 4.51 +/- 0.13 (8), respectively (P < 0.01). 3. PPADS at a concentration of 1 microM had no effect on contractions of the ear artery evoked by EFS (4-64 Hz; 1 microM phentolamine present).(ABSTRACT TRUNCATED AT 250 WORDS)     

Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C.

1. The effect of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), on human platelets has been investigated. 2. Okadaic acid exerts a general increase in phosphorylation of platelet proteins but did not induce aggregation or secretion of 5-hydroxytryptamine (5-HT). Okadaic acid, however, did inhibit thrombin-induced functional responses. 3. Maximally effective concentrations of prostacyclin, to elevate adenosine 3'-5'-cyclic monophosphate (cyclic AMP), or phorbol dibutyrate, to activate protein kinase C, inhibited the formation of inositol phosphates by thrombin by approximately 60%. When used in combination, prostacyclin and phorbol dibutyrate reduced the levels of inositol phosphates induced by thrombin to 11%. 4. Okadaic acid (1 microM) decreased thrombin-induced formation of inositol phosphates by approximately 55% and increased the inhibitory action of prostacyclin or phorbol dibutyrate. Okadaic acid had no further effect when prostacyclin and phorbol dibutyrate were used in combination. 5. These results suggest that protein kinases A and C act to inhibit phospholipase C by distinct mechanisms and that their action is reversed by PP1 and/or PP2A.     

Effects of cyclopiazonic acid on contractility and ecto-ATPase activity in guinea-pig urinary bladder and vas deferens.

1. Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic ATPase, was tested on guinea-pig urinary bladder and vas deferens for its ability: (1) to modify contractile responses to electrical field stimulation (EFS), exogenous ATP, alpha,beta-methylene ATP (alpha,beta-MeATP), carbachol, noradrenaline (NA), histamine, and KCl; (2) to affect ecto-ATPase activity; (3) to modify the release of ATP evoked by EFS. 2. In the urinary bladder, CPA (10 microM) potentiated contractile responses to EFS, exogenous ATP (100 microM), alpha,beta-meATP (1 microM), carbachol (0.5 microM), histamine (30 microM) and KCl (30 mM). In the vas deferens, CPA (10 microM) potentiated responses to EFS, ATP, alpha,beta-meATP, NA (100 microM) and KCl. CPA at a concentration of 1 microM had no effect on ATP-induced relaxation of carbachol-precontracted guinea-pig taenia coli, and at a concentration of 10 microM it markedly increased spontaneous contractile activity of taenia. 3. Ecto-ATPase was estimated to have Vmax and Km values of 0.98 nmol Pi 30 min-1 mg-1 wet tissue and 881 microM ATP in the urinary bladder, and 0.75 nmol Pi 30 min-1 mg-1 wet tissue and 914 microM ATP in the vas deferens, respectively. CPA at a concentration of 10 microM significantly inhibited ecto-ATPase activity by 18% in the urinary bladder and by 24% in the vas deferens. 4. In the guinea-pig vas deferens, CPA significantly potentiated ATP release evoked by EFS from 2.2 +/- 0.8 (6) pmol ATP min-1 g-1 wet tissue to 35.2 +/- 4.8 (6) pmol ATP min-1 g-1 wet tissue (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

The antinociceptive effect of morphine is reversed by okadaic acid in morphine-naive but not in morphine-tolerant mice

The activation of specific subtypes of serine/threonine protein phosphatases (PPs) plays a role in the antinociceptive effect of acute morphine, but it is not known whether these enzymes are involved in morphine-induced antinociception in morphine-tolerant animals. We evaluated the effects of both okadaic acid (a selective inhibitor of some serine/threonine PPs) and its inactive analogue L-norokadaone on the antinociception induced by morphine in morphine-naive and -tolerant female mice in the tail-flick test. Okadaic acid (0.01 and 1 pg/mouse, i.c.v.), but not L-norokadaone (1 pg/mouse, i.c.v.), antagonized in a dose-dependent way the antinociception induced by morphine (1-16 mg/kg, s.c.) in morphine-naive animals. However, both okadaic acid (0.01 and 1 pg/mouse, i.c.v.) and L-norokadaone (1 pg/mouse, i.c.v.) were unable to modify the antinociceptive effect of morphine in morphine-tolerant mice. These results suggest that in morphine-induced thermal analgesia, the role of serine/threonine PPs highly sensitive to okadaic acid is different in morphine-tolerant and morphine-naive female mice.     

Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells

Mast cells participate in allergies, and also in immunity and inflammation by secreting proinflammatory cytokines. Flavonoids are naturally occurring polyphenolic plant compounds, one group of which -- the flavonols, inhibits histamine and some cytokine release from rodent basophils and mast cells. However, the effect of flavonols on proinflammatory mediator release and their possible mechanism of action in human mast cells is not well defined. Human umbilical cord blood-derived cultured mast cells (hCBMCs) grown in the presence of stem cell factor (SCF) and interleukin (IL)-6 were preincubated for 15 min with the flavonols quercetin, kaempferol, myricetin and morin (0.01, 0.1, 1, 10 or 100 microM), followed by activation with anti-IgE. Secretion was quantitated for IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), histamine and tryptase levels. Release of IL-6, IL-8 and TNF-alpha was inhibited by 82-93% at 100 microM quercetin and kaempferol, and 31-70% by myricetin and morin. Tryptase release was inhibited by 79-96% at 100 microM quercetin, kampferol and myricetin, but only 39% by morin; histamine release was inhibited 52-77% by the first three flavonols, but only 28% by morin. These flavonols suppressed intracellular calcium ion elevations in a dose-response manner, with morin being the weakest; they also inhibited phosphorylation of the calcium-insensitive protein kinase C theta (PKC theta). Flavonol inhibition of IgE-mediated proinflammatory mediator release from hCBMCs may be due to inhibition of intracellular calcium influx and PKC theta signaling. Flavonols may therefore be suitable for the treatment of allergic and inflammatory diseases.     

Endothelin-induced contraction and mediator release in human bronchus.

1. To elucidate the role of acetylcholine and various autacoids in endothelin-1 (ET-1)-induced contraction in human bronchus, the effects of various receptor antagonists were examined. In addition, the ability of ET-1 to stimulate the release of histamine, peptidoleukotrienes and prostanoids was determined. 2. ET-1 was a potent and effective contractile agonist in human bronchus, possessing similar potency and efficacy to leukotriene D4 (LTD4); EC50 (-log M): ET-1 = 7.76 +/- 0.09, n = 7; LTD4 = 8.46 +/- 0.53, n = 7; P > 0.2; maximum response (% 10 microM pre-carbachol): ET-1 = 103.8 +/- 17.4, n = 7; LTD4 = 95.5 +/- 9.3, n = 7; P > 0.6. 3. The cyclo-oxygenase inhibitor, sodium meclofenamate (1 microM) or the potent and selective thromboxane receptor antagonist, SQ 29,548 (1 microM) were without significant effect on ET-1 concentration-response curves. 4. In the presence of sodium meclofenamate (1 microM), the muscarinic receptor antagonist, atropine (1 microM), the platelet activating factor (PAF) receptor antagonist, WEB 2086 (1 microM) or the combination of the H1-histamine receptor antagonist, mepyramine (10 microM) and the leukotriene receptor antagonist, SK&F 104353 (10 microM), were without marked effect on ET-1 concentration-response curves. In addition, the combination of all four receptor antagonists did not antagonize ET-1-induced contraction. 5. ET-1 (0.3 microM) did not stimulate the release of histamine or immunoreactive leukotrienes from human bronchus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Display of the characteristics of endothelium-derived hyperpolarizing factor by a cytochrome P450-derived arachidonic acid metabolite in the coronary microcirculation.

1. In addition to nitric oxide (NO) and prostacyclin (PGI2) an endothelium-derived factor, which hyperpolarizes vascular smooth muscle cells via activation of K+ channels, contributes to the dilator effect of bradykinin in different vascular beds. Since this so-called endothelium-derived hyperpolarizing factor (EDHF) also seems to play an important role in the coronary circulation, we investigated its nature and mechanism of action in the rat isolated perfused heart (Langendorff preparation). 2. Bolus injections of bradykinin (1, 10, and 100 pmol) elicited a transient dose-dependent dilator response (e.g., 12 +/- 2% decrease in coronary perfusion pressure (CPP) at 10 pmol bradykinin, n = 41). Administration of the cyclo-oxygenase inhibitor, diclofenac (1 microM), augmented the bradykinin-induced dilation approximately twofold (n = 9 P < 0.01). Combined treatment with the NO synthase inhibitor, NG-nitro-L-arginine (30 microM) and diclofenac (1 microM) significantly reduced the duration, but increased the amplitude of the dilator response to bradykinin (27 +/- 2% decrease in CPP, n = 24, P < 0.01). 3. The abolition of this NG-nitro-L-arginine/diclofenac-insensitive dilator response to bradykinin by tetrabutylammonium (0.3 mM), an inhibitor of Ca(2+)-dependent K+ channels (4 +/- 1% decrease in CPP, n = 6, P < 0.01), supports the view that the dilator compound released in the coronary microcirculation is EDHF. 4. This EDHF-type dilation was reversibly inhibited by the phospholipase A2 inhibitor, quinacrine (3 microM, 9 +/- 3% decrease in CPP, n = 6, P < 0.01) and by the cytochrome P450 inhibitor SKF525a (3 microM, 6 +/- 1% decrease in CPP, n = 6, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

The relaxant effect of okadaic acid on canine basilar artery involves activation of PKCalpha and phosphorylation of the myosin light chain at Thr-9

Vasodilator responses induced by okadaic acid were investigated in canine basilar artery precontracted with 80 mM KCl. Okadaic acid (1 microM) relaxed the artery and this relaxant effect was partially inhibited by G?6976, a conventional protein kinase C inhibitor, and calphostin C, an inhibitor of conventional and novel PKCs. Rottlerin, a specific inhibitor of PKCdelta, did not influence okadaic acid's effect. KCl increased phosphorylation of 20,000-Dalton myosin light chain (MLC(20)) at Ser-19. Okadaic acid additionally increased MLC(20) phosphorylation at Thr-18 and Thr-9, resulting in triphosphorylation of MLC(20). This phosphorylation was inhibited by G?6976. Okadaic acid stimulated phosphorylation of PKCalpha and 17,000-Dalton PKC-potentiated inhibitory phosphoprotein (CPI-17), and G?6976 inhibited these phosphorylations. These results suggest that okadaic acid's relaxant effect involves MLC(20) triphosphorylation through a direct phosphorylation by PKCalpha and an indirect phosphorylation by inhibition of myosin light chain phosphatase through PKCalpha-mediated CPI-17 phosphorylation.     

The histamine H1 receptor in GT1-7 neuronal cells is regulated by calcium influx and KN-62, a putative inhibitor of calcium/calmodulin protein kinase II.

1. In GT1-7 cells, histamine stimulated the initial [Ca2+]i transient in a dose-dependent manner with a best-fit EC50 value of 4.2 +/- 4.2 microM (mean +/- s.e.mean, n = 4) and a best-fit maximal effect of 138 +/- 56 nM (n = 4) increase above basal calcium levels. 2. Pretreatment of cells with 30 microM histamine for 30 min desensitized the population mean peak calcium signal by 53% to 75 +/- 9 nM, (n = 3, P < 0.04). Analysis of the individual cells revealed that 39 +/- 7% (n = 94 cells from 8 experiments) of pretreated cells exhibited desensitized histamine-stimulated [Ca2+]i transients of < or = 1 standard deviation below the control cells mean calcium transient level. 3. The desensitization induced by histamine was prevented (P < 0.01) by KN-62 (10 microM), a putative inhibitor of the calcium/calmodulin-dependent protein kinase II (CaMKII). KN-62 (10 microM) alone did not induce [Ca2+]i mobilization, nor did it antagonize the histamine-stimulated [Ca2+]i signal. In addition, KN-62 did not appear to have its effect by hastening the rate of recovery from desensitization. 4. Histamine pretreatment in nominal (zero calcium + 0.2 mM EGTA) or in low (0.3 mM) extracellular calcium did not induce histamine receptor desensitization, supporting a role for extracellular calcium in the homologous H1 receptor desensitization process. 5. Histamine (30 microM) stimulated at least four different types of [Ca2+]i signals in GT1-7 cells. The majority (61%) were of single spikes with the remaining cells showing some form of calcium oscillatory behaviour. The proportion of GT1-7 cells showing histamine-induced calcium oscillations was histamine concentration-dependent and significantly reduced after acute desensitization. KN-62, when present during histamine pretreatment, prevented this fall in calcium oscillation. Under the conditions of nominal or 0.3 mM extracellular calcium the proportion of cells exhibiting histamine-stimulated calcium oscillations was not significantly different from the controls. 6. Bradykinin stimulated a [Ca2+]i transient in GT1-7 cells with a population mean peak response of 147 +/- 8 nM (n = 5) over basal levels. The bradykinin-induced [Ca2+]i signal was without any calcium oscillatory activity. Histamine pretreatment caused the heterologous desensitization of the bradykinin [Ca2+]i signal (44% reduction, P < 0.007), which was unaffected by KN-62. 7. The results presented here suggest that the histamine-mediated homologous H1 receptor desensitization process involves extracellular calcium and can be blocked by KN-62, a putative inhibitor of CaMKII. In contrast, KN-62 does not appear to prevent the histamine-mediated heterologous desensitization cascade. These findings suggest fundamental differences in the mechanisms underlying homologous and heterologous H1 receptor desensitization pathways in GT1-7 neuronal cells.     

Role for intracellular platelet-activating factor in the circulatory failure in a model of gram-positive shock.

1. This study investigates the effects of two structurally different antagonists of platelet-activating factor (PAF), BN52021 and WEB2086, on the circulatory and renal failure elicited by lipoteichoic acid (LTA) from Staphylococcus aureus (an organism without endotoxin) in anaesthetized rats. 2. Administration of LTA (10 mg kg-1, i.v.) caused hypotension and vascular hyporeactivity to noradrenaline (1 microgram kg-1, i.v.) WEB2086 (5 mg kg-1, i.v., 20 min before and 150 min after LTA) inhibited the delayed fall in mean arterial blood pressure (at 300 min: 99 +/- 6 mmHg vs. 75 +/- 6 mmHg, P < 0.01) and prevented the decrease in pressor response to noradrenaline (at 300 min: 36 +/- 5 mmHg min vs. 17 +/- 5 mmHg min, P < 0.01). Surprisingly, BN52021 (20 mg kg-1, i.v., 20 min before and 150 min after LTA) neither prevented the hypotension (74 +/- 6 mmHg) nor the vascular hyporeactivity (21 +/- 5 mmHg min). However, BN52021 inhibited the hypotension to injections of PAF as well as the circulatory failure elicited by lipopolysaccharides (10 mg kg-1, i.v.). 3. LTA caused an increase in plasma concentration of creatinine from 39 +/- 5 microM (sham-operated) to 70 +/- 8 microM and urea from 4.7 +/- 0.1 to 13.1 +/- 1.6 mM. The renal failure elicited by LTA was significantly inhibited by WEB2086 (creatinine: 45 +/- 4 microM and urea: 5.7 +/- 0.7 mM), but not by BN52021. 4. The induction of nitric oxide synthase activity in lungs by LTA was attenuated by WEB2086 from 98 +/- 17 to 40 +/- 15 pmol L-citrulline 30 min-1 mg-1 protein (P < 0.01), but not by BN52021 (148 +/- 21 pmol L-citrulline 30 min-1 mg-1 protein). Similarly, WEB2086, but not BN52021, inhibited the increase in plasma nitrite concentration associated with the delayed circulatory failure caused by LTA. The release of tumour necrosis factor-alpha (TNF-alpha) after injection of LTA was not attenuated by WEB2086. 5. The induction of nitrite release by cultured macrophages activated with LTA (10 micrograms ml-1 for 24 h) was inhibited by 74 +/- 4% by WEB2086 (3 x 10(-4) M), but not by BN52021, indicating that only WEB2086 acts on intracellular PAF receptors. 6. Thus, the intracellular release of PAF contributes to the circulatory and renal failure and induction of nitric oxide synthase elicited by LTA in anaesthetized rats. The difference between the two structurally different PAF antagonists in our septic shock models using either LTA or lipopolysaccharide (LPS), shows the importance of models for Gram-positive sepsis in the elucidation of the pathophysiology of septic shock and for the evaluation of potential drugs.     

Effect of inhibitors of transmethylation on histamine release from human basophils

Methylation reactions mediated by $\text{S-}\text{adenosyl-}\text{l}\text{-methionine}$ (AdoMet) play an important role in a number of biological reactions including bacterial and human monocyte chemotaxis. This study evaluated the role of methylation reactions in histamine release from human basophils. Methylation was blocked by several methods. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an inhibitor of adenosine deaminase (EC 3.5.4.4), blocked the IgE-mediated histamine release with an ic₅₀ (concentration of drug required to produce 50 per cent inhibition) of 0.33 mM. Preincubation of leukocytes with adenosine caused some inhibition of IgE-mediated histamine release. This inhibition by adenosine was potentiated by the addition of 1 × 10^?5 M EHNA which alone did not affect histamine release. Further addition of l-homocysteine thiolactone at 1 × 10^?4 M potentiated the inhibitory effect of EHNA plus adenosine. The effect of l-homocysteine thiolactone was dose dependent. 3-Deazaadenosine (DZA), an inhibitor of $\text{S-}\text{adenosyl-}\text{l}\text{-homocysteine}$ hydrolase (EC 3.3.1.1) inhibited IgE-initiated histamine release from human basophils with an ic₅₀ of ~1 mM. This inhibition was potentiated by l-homocycsteine thiolactone. The inhibition by DZA was not potentiated by two different phospho-diesterase inhibitors suggesting that the action of DZA was not through changes in intracellular levels of cyclic AMP. DZA inhibited during the Ca²⁺-independent activation step of IgE-mediated basophil histamine release. In contrast, when histamine release was induced by the calcium ionophore A23187, f'Met-Leu-Phe or zymosan-activated serum, there was either no inhibition or enhancement of histamine release by these inhibitors of transmethylation. These results suggest that AdoMet-mediated methylation plays an important role in IgE-initiated histamine secretion from human basophils and that release induced by A23187, fMet-Leu-Phe, or C5A, bypasses this reaction step.     

Inhibition of excitatory non-adrenergic non-cholinergic bronchoconstriction in guinea-pig airways in vitro by activation of an atypical 5-HT receptor.

1. The effect of 5-hydroxytryptamine (5-HT) was studied on excitatory neurally mediated non-adrenergic non-cholinergic (NANC) contractions evoked by electrical field stimulation (EFS) in guinea-pig isolated bronchi. 2. 5-HT (0.1-100 microM) produced a concentration-dependent inhibition of the excitatory NANC response with 50.9 +/- 5.0% (n = 5, P < 0.01) inhibition at 100 microM. This inhibition was not significantly affected by the 5-HT2 antagonist, ketanserin (1 microM) when inhibitions (+/- ketanserin) at each concentration of 5-HT were compared by unpaired t tests; however, this concentration appeared to produce a leftward shift (approximately 10 fold) of the 5-HT concentration-inhibition curve. Ketanserin (1 microM) was effective in blocking bronchoconstriction evoked by activation of 5-HT2A receptors on airway smooth muscle. In the presence of ketanserin (1 microM) 5-HT (100 microM) evoked an inhibition of 57.4 +/- 5.9% (n = 5, P < 0.01) with an EC50 of 0.57 microM. 3. Inhibition evoked by 5-HT (0.1-100 microM) was unaffected by the alpha-adrenoceptor antagonist phentolamine (1 microM), the beta 2-adrenoceptor antagonist, ICI 118551 (0.1 microM), the 5-HT1A/B antagonist, cyanopindolol (1 microM) or the 5-HT3/4 antagonist, ICS 205-930 (1 microM). 4. Methiothepin (0.1 microM) produced an insurmountable inhibition of the effect of 5-HT (0.1-100 microM), reducing the maximum inhibition produced by 5-HT (100 microM) to 30.2 +/- 5.0% (n = 5, P < 0.001) and suggesting a non-competitive antagonism. Methiothepin inhibited the effect of 5-HT (10 microM) in a concentration-dependent manner with an IC50 of 81 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein phosphatase activity is increased in a rat model of long-term β-adrenergic stimulation

We tested the hypothesis that altered phosphorylation of Ca2+ regulatory proteins contributes to contractile anomalies in cardiac hypertrophy. Cardiac hypertrophy was induced in rats by chronic s.c. administration of isoproterenol (Iso, 2.4 mg/kg/day) via osmotic minipumps. On day 2 of Iso treatment the expression of atrial natriuretic factor was increased, time of relaxation in isolated papillary muscles shortened and protein expression of phospholamban (PLB) and sarcoplasmic reticulum Ca2+-ATPase reduced. In addition, the phosphorylation state of PLB at serine-16 and threonine-17 was decreased from (arbitrary units) 2.3+/-0.3 to 1.1+/-0.2 and from 4.1+/-0.6 to 2.1+/-0.2, respectively. This was not accompanied by altered activity of PLB-phosphorylating protein kinases (protein kinase A or Ca2+/calmodulin-dependent protein kinase II), whereas the activity of types 1 and 2A protein phosphatases (PP1 and -2A respectively) was enhanced from 1.1+/-0.08 to 1.71+/-0.13 nmol/mg/min. Iso treatment did not alter the PP1/PP2A activity ratio and 1 nmol/l okadaic acid, a concentration which completely blocks the catalytic subunit of PP2A, inhibited about 40% of total PP activity in all groups studied. These data indicate that the activity of both PP1 and PP2A were increased. All effects of Iso treatment were abolished by co-administration of propranolol (29.7 mg/kg/day). It is concluded that dephosphorylation of PLB is due to enhanced activity of PP1 and PP2A. We suggest that chronic beta-adrenergic stimulation, which occurs in human cardiac hypertrophy and failure, can lead to increased activity of PPs. This may contribute to altered contractile responses in the hypertrophied heart.