Stretch-induced triphosphorylation of myosin light chain and myogenic tone in canine basilar artery

The relationship between phosphorylation of 20,000 Da myosin light chain (MLC20) and contraction in response to mechanical stretch was investigated in the canine basilar artery. A slow stretch (at a rate of 1 mm/s and a stimulus period for 15 min) increased triphosphorylated MLC20 despite lowered intracellular calcium concentration and mechanical activities, such as myogenic tone, shortening velocity and stiffness of the artery. Nicardipine, a Ca2+ channel blocker, and ML-9, a myosin light chain kinase (MLCK) inhibitor, partially inhibited the stretch-induced MLC20 phosphorylation. The remained phosphorylation was further reduced by calphostin C, a protein kinase C (PKC) inhibitor. Y-27632, a Rho-kinase inhibitor, inhibited phosphorylation of myosin light chain phosphatase and attenuated MLC20 phosphorylation. These results suggest that slow stretch induces triphosphorylation of MLC20, which is mediated by MLCK, PKC, and Rho-kinase, and that the triphosphorylation of MLC20 does not result in myogenic contraction, rather seems to counteract it.     

A role for Rho-kinase in Ca-independent contractions induced by phorbol-12,13-dibutyrate

1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca(2+)-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca(2+)-free solution, 0.1 and 1 micromol/L PDBu induced contraction and myosin light chain (MLC(20)) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 micromol/L), an inhibitor of Rho-kinase, but not ML7 (10 micromol/L), an inhibitor of myosin light chain kinase, inhibited Ca(2+)-independent contractions induced by PDBu. 3. In Ca(2+)-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 micromol/L Ro31-8220, an inhibitor of PKC. 4. In Ca(2+)-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca(2+)-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.     

Inhibition of protein kinase Calpha enhances anticancer agent-induced loss of anchorage-independent growth regardless of protection against apoptosis by Bcl-2

In the present study, we investigated the effects of several selective protein kinase C (PKC) inhibitors (G?6976, G?6983, bisindolylmaleimide I, and rottlerin) in combination with conventional anticancer drugs on apoptosis and long-term anchorage-independent growth of both parental and Bcl-2-overexpressing mammary adenocarcinoma MTLn3 cells. In normal MTLn3 cells, doxorubicin- and etoposide-induced apoptosis was not affected by any of the PKC inhibitors. However, Bcl-2-mediated cytoprotection against apoptosis was slightly counteracted by G?6976, a selective inhibitor of PKCalpha, as well as by transient overexpression of dominant-negative PKCalpha. Doxorubicin and etoposide both inhibited anchorage-independent growth; for doxorubicin, this occurred at concentrations that did not yet cause apoptosis. Overexpression of Bcl-2 did not overcome these growth-inhibitory effects. The effects of doxorubicin on colony formation were potentiated by G?6976, G?6983, and bisindolylmaleimide I but not rottlerin. In contrast, etoposide-induced loss of clonogenicity was primarily enhanced by G?6976. G?6976 alone, but not G?6983, bisindolylmaleimide I, or rottlerin, inhibited colony formation in soft agar. This effect of G?6976 correlated with inhibition of cell cycle progression. Overall, the data indicate that pharmacological inhibitors of PKCalpha in combination with anticancer drugs, act additively to inhibit long-term anchorage-independent tumor cell growth, independent of apoptosis induction. Importantly, similar additive effects are observed in Bcl-2 overexpressing cells.     

Mechanism of potentiation by tea epigallocatechin of contraction in porcine coronary artery: the role of protein kinase Cδ-mediated CPI-17 phosphorylation

The effects of green tea catechins, (+)- and (−)-catechins (C), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCG), on vascular contractility were investigated in porcine coronary artery. At the concentration of 200 μM, only EGC, but not other catechins, potentiated high K⁺-induced contraction in a concentration-dependent manner, although EGC by itself did not produce contraction. The potentiator effect of EGC was still observed in endothelium-denuded preparations. Moreover, EGC increased the translocation of protein kinase Cδ (PKCδ) from the cytosol to the plasma membrane and increased phosphorylations of 17-kDa PKC-potentiated protein phosphatase inhibitor protein (CPI-17) and myosin light chain (MLC₂₀). These effects of EGC were inhibited by the PKCδ inhibitor rottlerin, but not by the conventional PKC inhibitor Gö6976. These results suggest that EGC activates PKCδ, leading to the phosphorylation of CPI-17, which in turn inhibits myosin light chain phosphatase and increases MLC₂₀ phosphorylation. The series of events would increase Ca²⁺ sensitivity of contractile elements, thereby augmenting high K⁺-induced vascular contraction.     

Okadaic acid enhances human T cell activation and phosphorylation of an internal substrate induced by phorbol myristate acetate.

Okadaic acid is a potent tumor promoter and an inhibitor of serine/threonine-specific protein phosphatases. We studied the effect of okadaic acid in human T cell activation and phosphorylation of internal substrates. Okadaic acid at up to 4 nM enhanced phorbol myristate acetate (PMA)-induced proliferation and CD25 (IL-2 receptor, p55) expression, although it showed no activation by itself. Okadaic acid induced hyperphosphorylation of a 60 kDa protein in T cells as well as non-T cells, as reported in fibroblasts and keratinocytes. Preincubation with 4 nM okadaic acid enhanced PMA induced phosphorylation of the 80 kDa protein, an internal substrate of protein kinase C in T cells. These results suggest that okadaic acid inhibited dephosphorylation of protein kinase C specific substrates, and as a result, enhanced T cell activation mediated by protein kinase C pathway.     

Okadaic acid increases the phosphorylation state of alpha1A-adrenoceptors and induces receptor desensitization

Okadaic acid, a protein phosphatase inhibitor, and phorbol myristate acetate, an activator of protein kinase C, increased the phosphorylation state of alpha1A-adrenergic receptors. The effects of these agents were of similar magnitude but that of okadaic acid developed more slowly. Wortmannin (inhibitor of phosphoinositide 3-kinase), but not staurosporine (inhibitor of protein kinase C), abolished the effect of okadaic acid on the alpha1A-adrenoceptor phosphorylation state. The effect of phorbol myristate acetate on this parameter was blocked by staurosporine and only partially inhibited by wortmannin. Okadaic acid markedly increased the co-immunoprecipitation of both the catalytic and regulatory subunits of phosphatidylinositol 3-kinase and of Akt/protein kinase B with the adrenoceptor and only marginally increases receptor association with protein kinase C epsilon. Okadaic acid induced desensitization of alpha1A-adrenoceptors as evidenced by a decreased ability of noradrenaline to increase intracellular calcium. Such desensitization was fully reverted by wortmannin. Our data indicate that inhibition of serine/threonine protein phosphatases increases the phosphorylation state of alpha1A-adrenergic receptor and alters the adrenoceptor function.     

Endothelin-1-induced phosphorylation of the 20-kDa myosin light chain and caldesmon in porcine coronary artery smooth muscle.

Endothelin-1 (ET), a potent vasoconstrictor, induces a sustained increase in the phosphorylation level of the 20-kDa myosin light chain (MLC) in porcine coronary artery strips. ET also induces late phosphorylation of caldesmon, which is mimicked by 12-deoxyphorbol 13-isobutyrate, but not by 60 mM KCl. Nitroglycerin, a vasorelaxant, completely reverses the ET-induced phosphorylation of MLC, but not that of caldesmon. These results suggest an important regulatory role of MLC phosphorylation in ET-induced contraction.     

Capsaicin-induced, capsazepine-insensitive relaxation of the guinea-pig ileum

The mechanisms underlying transient receptor potential vanilloid receptor type 1 (TRPV1)-independent relaxation elicited by capsaicin were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC(20)) in ileum longitudinal smooth muscles of guinea-pigs. In acetylcholine-stimulated tissues, capsaicin (1-100 microM) and resiniferatoxin (10 nM-1 microM) produced a concentration-dependent relaxation. The relaxant response was attenuated by 4-aminopyridine and high-KCl solution, but not by capsazepine, tetraethylammonium, Ba(2+), glibenclamide, charybdotoxin plus apamin nor antagonists of cannabinoid receptor type 1 and calcitonin-gene related peptide. A RhoA kinase inhibitor reduced the relaxant effect of capsaicin at 30 microM. Capsaicin and resiniferatoxin reduced acetylcholine- and caffeine-induced transient contractions in a Ca(2+)-free, EGTA solution. Capsaicin at 30 microM for 20 min did not alter basal levels of MLC(20) phosphorylation, but abolished an increase by acetylcholine in MLC(20) phosphorylation. It is suggested that the relaxant effect of capsaicin at concentrations used is not mediated by TRPV1, but by 4-aminopyridine-sensitive K(+) channels, and that capsaicin inhibits contractile mechanisms involving Ca(2+) release from intracellular storage sites. The relaxation could be explained by a decrease in phosphorylation of MLC(20).     

Effects of NA0344, a new smooth muscle relaxant, on the actin-myosin-ATP interaction and myosin light chain phosphorylation in vitro

1. Effects of antibiotic NA0344, a smooth muscle relaxant, on phosphorylation of myosin light chain (MLC, 20 kDa) were compared with those on actin-myosin-ATP interaction using native actomyosin preparation containing MLC kinase activity. 2. MLC kinase was shown to be the site of action of NA0344. 3. NA0344 inhibited the interaction and phosphorylation with IC50 = 7.5 x 10(-6) and 1.6 x 10(-5) M, respectively. 4. The discrepancy between the inhibitory effects is explained that myosin is in an active form when myosin is fully phosphorylated. 5. However, the inhibitory effects of NA0359 and NA0362, analogs of NA0344, on the phosphorylation were similar to or more effective than those on the interaction, which cannot be explained by the active form hypothesis. 6. Plausible explanations for the discrepancies are discussed.     

Rho-kinase inhibitor inhibits both myosin phosphorylation-dependent and -independent enhancement of myofilament Ca2+ sensitivity in the bovine middle cerebral artery

The role of Rho kinase in Ca2+ sensitization of the contractile apparatus in smooth muscle was investigated in the bovine middle cerebral artery. U46619, a thromboxane A2 analog, induced a greater sustained contraction with a smaller [Ca2+]i elevation than that seen with 118 mm K+. The level of myosin light chain (MLC) phosphorylation obtained in the initial phase of the contraction was higher than that seen with 118 mm K+; thereafter, it gradually declined to a comparable level in the late phase. During the steady state of the U46619-induced contraction, Y27632 (10 microM), a Rho-kinase inhibitor, partially inhibited [Ca2+]i, although it substantially inhibited tension and MLC phosphorylation. Wortmannin (10 microM), an MLC kinase inhibitor, had no significant effect on [Ca2+]i, but it completely inhibited MLC phosphorylation and partially inhibited tension. The wortmannin-resistant tension development was thus not associated with MLC phosphorylation, and this component was completely inhibited by Y27632. In conclusion, U46619 enhanced Ca2+ sensitivity in a manner both dependent and independent of MLC phosphorylation in the bovine middle cerebral artery. Both mechanisms of Ca2+ sensitization can be inhibited by the Rho-kinase inhibitor.     

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.     

Characterization of capsaicin-induced, capsazepine-insensitive relaxation of ileal smooth muscle of rats

The mechanisms underlying the capsaicin-induced relaxation of the acetylcholine- as well as KCl-contraction were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC(20)) in ileal longitudinal smooth muscles of rats. Capsaicin relaxed acetylcholine- and KCl-stimulated preparations in a concentration-dependent manner; the former was less sensitive to capsaicin than the latter and maximum responses to capsaicin (a percentage of papaverine-induced relaxation) were 70.6+/-7.5%, n=10 and 97.1+/-0.9%, n=13, P<0.05, respectively. The response showed no desensitization. Like nifedipine, capsaicin relaxed the tissue precontracted with an agonist of L-type Ca(2+) channels as well. The relaxant effect of capsaicin was not inhibited by capsazepine (a selective antagonist of vanilloid VR1 receptors), nitro-l-arginine, indomethacin, guanethidine, nor by inhibitors of soluble guanylate cyclase. Capsaicin inhibited acetylcholine-induced transient contraction in a Ca(2+)-free, EGTA solution. Phosphorylation of MLC(20) (a percentage of phosphorylated to total MLC(20)) was increased 1 min after application of 10 microM acetylcholine (7.8+/-2.0%, n=6 vs. 22.6+/-3.2%, n=6) and of 65.9 mM KCl (2.2+/-0.3%, n=8 vs. 10.7+/-1.7%, n=12). Capsaicin reduced the KCl-induced increase more markedly than acetylcholine-induced increase in MLC(20) phosphorylation. When the tissue was contracted for 20 min with acetylcholine, MLC(20) phosphorylation was increased, and capsaicin reduced markedly the contraction and abolished MLC(20) phosphorylation both elicited by acetylcholine. It is suggested that capsaicin relaxes the rat ileum via its direct action on smooth muscle, and that capsaicin inhibits contractile mechanisms involving extracellular Ca(2+) influx via non-L-type Ca(2+) channels, possibly via store-operated Ca(2+) channels and Ca(2+) release from intracellular storage sites. The effects of capsaicin on acetylcholine- and KCl-induced contraction could be explained by a decrease in MLC(20) phosphorylation.     

Augmentation of endothelin-1-induced phosphorylation of CPI-17 and myosin light chain in bronchial smooth muscle from airway hyperresponsive rats

Airway hyperresponsiveness (AHR) associated with heightened airway resistance and inflammation is a characteristic feature of bronchial asthma. It has been demonstrated that contraclile responsiveness to endothelin-1 (ET-1) in repeated antigen challenge-induced airway hyperresponsive bronchial preparation was significantly increased. ET-1 is a potent contracting substance for various smooth muscles including airways. In addition to the classical Ca(2+)-mediated contraction, ET-1 also induced Ca(2+) sensitization of contraction. However, it is not clear whether ET-1 stimulation also activates the CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) pathway in airway smooth muscles. Therefore, the changes in ET-1-induced activation/phosphorylation of CPI-17 and myosin light chain (MLC) in bronchial smooth muscle of repeatedly antigen-challenged rats were examined. The levels of ET-1-induced phosphorylation of CPI-17 and MLC were increased much more markedly in the AHR group than in the sensitized control animals. It might be suggested that the augmented activation of CPI-17 observed in the hyperresponsive bronchial smooth muscle is responsible for the enhanced agonists-induced contraction of bronchial smooth muscle in AHR rats.     

Dimethyl sulphoxide relaxes rabbit detrusor muscle by decreasing the Ca2+ sensitivity of the contractile apparatus

BACKGROUND AND PURPOSE: The intravesical administration of dimethyl sulphoxide (DMSO) is used to alleviate the symptoms of interstitial cystitis. We investigated the relaxant effect of DMSO and its underlying mechanism in the detrusor muscle. EXPERIMENTAL APPROACH: The effects of DMSO on contraction, on Ca2+ sensitivity of myofilaments, and on myosin light chain (MLC) phosphorylation were investigated in both intact and alpha-toxin-permeabilized strips of rabbit detrusor muscle. KEY RESULTS: In fura-PE3-loaded strips, DMSO (>1%) induced a significant relaxation during sustained contractions induced by 60 mM K+-depolarization or 10 microM carbachol, while having no effect on the [Ca2+](i) level. DMSO decreased the level of MLC phosphorylation during the contractions induced by 60 mM K+ and 10 microM carbachol. DMSO also inhibited both the contraction and MLC phosphorylation induced by calyculin-A in intact strips. In the alpha-toxin-permeabilized preparations, DMSO relaxed the Ca2+-induced contraction and also inhibited the tension development induced by a stepwise increment of Ca2+ concentrations. Such a relaxant effect of DMSO was enhanced in the presence of phosphate. CONCLUSIONS AND IMPLICATIONS: DMSO relaxes rabbit detrusor muscle by decreasing the Ca2+ sensitivity of myofilaments. Inhibition of the kinase activities involved in myosin phosphorylation may play a major role in DMSO-induced Ca2+ desensitization. Inhibition of the cross-bridge cycling at the step of phosphate release may also contribute to the relaxant effect of DMSO. Such relaxant effects of DMSO could be linked to the therapeutic effect of DMSO in interstitial cystitis.     

c-Jun N-terminal kinase contributes to norepinephrine-induced contraction through phosphorylation of caldesmon in rat aortic smooth muscle

Vascular smooth muscle contraction is mediated by activation of extracellular signal-regulated kinase (ERK) 1/2, an isoform of mitogen-activated protein kinase (MAPK). However, the role of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) in vascular smooth muscle contraction has not been defined. We investigated the role of JNK in the contractile response to norepinephrine (NE) in rat aortic smooth muscle. NE evoked contraction in a dose-dependent manner, and this effect was inhibited by the JNK inhibitor SP600125. NE increased the phosphorylation of JNK, which was greater in aortic smooth muscle from hypertensive rats than from normotensive rats. NE-induced JNK phosphorylation was significantly inhibited by SP600125 and the conventional-type PKC (cPKC) inhibitor G?6976, but not by the Rho kinase inhibitor Y27632 or the phosphatidylinositol 3-kinase inhibitor LY294002. Thymeleatoxin, a selective activator of cPKC, increased JNK phosphorylation, which was inhibited by G?6976. SP600125 attenuated the phosphorylation of caldesmon, an actin-binding protein whose phosphorylation is increased by NE. These results show that JNK contributes to NE-mediated contraction through phosphorylation of caldesmon in rat aortic smooth muscle, and that this effect is regulated by the PKC pathway, especially cPKC.     

Protein kinase Calpha-dependent increase in Ca2+-independent phospholipase A2 in membranes and arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells

We previously reported that zymosan-stimulated, protein kinase C (PKC)-dependent arachidonic acid liberation occurs with association of Ca2+-independent phospholipase A2 (iPLA2) with the membranes of macrophage-like P388D1 cells. In the present study, the possible involvement of PKC isoforms (alpha, beta, delta, and epsilon) on the increase in iPLA2 was examined. Stimulation of P388D1 cells with zymosan induced increases in iPLA2 activity and protein in the membranes and liberation of arachidonic acid. In the stimulated cells, PKCalpha, PKCdelta, and PKCepsilon, but not PKCbeta, were increased in the membranes. The zymosan-induced increase in iPLA2 activity was suppressed by pretreatment with 4beta-phorbol 12-myristate 13-acetate for 10 hr, by which PKCalpha and PKCdelta, but not PKCbeta and PKCepsilon, were depleted, and by G?6976, a PKCalpha inhibitor, but not rottlerin, a PKCdelta inhibitor. The zymosan-induced release of arachidonic acid was also reduced by the PKC depletion and G?6976. However, stimulation with 4beta-phorbol 12-myristate 13-acetate alone did not increase iPLA2 activity in the membranes. Furthermore, the depletion of intracellular Ca2+ also impaired the zymosan-induced increase in iPLA2 activity in the membranes. However, no increase in iPLA2 activity was observed upon stimulation with Ca2+-mobilizing agents (ionomycin or thapsigargin). Cytochalasin D, an inhibitor of actin polymerization, suppressed the zymosan-induced increases in iPLA2 activity and protein in the membranes and the release of arachidonic acid. These results suggest that zymosan stimulates an increase in iPLA2 in the membranes of P388D1 cells probably through activation of PKCalpha in concert with cytochalasin D-sensitive events.     

Mechanism of human urotensin II-induced contraction in rat aorta

Urotensin II induced sustained contraction with an EC(50) value of 2.29 +/- 0.12 nM in rat aorta. Urotensin II (100 nM) transiently increased cytosolic Ca(2+) level ([Ca(2+)](i)), followed by a small sustained phase superimposed with rhythmic oscillatory change. In the presence of verapamil and La(3+), the [Ca(2+)](i) oscillation was completely inhibited, although a small transient increase in [Ca(2+)](i) remained. The urotensin II-induced contraction was also partially inhibited by verapamil and La(3+). Combined application of verapamil, La(3+), and thapsigargin completely inhibited the increase in [Ca(2+)](i) with only partial inhibition of the contraction elicited by urotensin II. Urotensin II increased myosin light chain (MLC) phosphorylation to a level greater than that induced by 72.7 mM KCl (high K(+)). Pretreatment with Go6983 (PKC inhibitor), U0126 (MEK inhibitor), or SB203580 (p38MARK inhibitor) partially inhibited the urotensin II-induced contraction with no effects on the high K(+)-induced contractions. Wortmannin (MLC kinase inhibitor) only partially inhibited urotensin II-induced contraction, although it completely inhibited the high K(+)-induced contraction. These results suggest that urotensin II-induced contraction is mediated by the Ca(2+)/calmodulin/MLC kinase system and modulated by the Ca(2+) sensitization mechanisms to increase MLC phosphorylation. In addition, activations of PKC, p38MAPK, and ERK1/2 modulate the contractility mediated by urotensin II in rat aorta.     

Regulation of human basophil function by phosphatase inhibitors.

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-mediated release of the pre-formed mediator, histamine from human basophils in a time- and dose-dependent manner. Optimal inhibition (77 +/- 4%, P < 0.0001) of histamine release was observed following a 2 h incubation with 1 microM okadaic acid. 2. Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE-dependent release of histamine. Concentrations required to inhibit release by 50% (IC50) were 0.6 microM for okadaic acid and 7.5 microM for okadaol, whereas okadaone was inactive. 3. The structurally-unrelated PP inhibitor, calyculin A, also inhibited IgE-dependent histamine release from basophils dose-dependently and was approximately six fold more potent than okadaic acid. 4. The IgE-mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 microM) > okadaol (3 microM) > okadaone (inactive). 5. Okadaic acid, okadaol and okadaone (all at 3 microM) inhibited the IgE-mediated generation of the cytokine interleukin 4 (IL4) from human basophils by 67 +/- 9% (P < 0.002), 48 +/- 14% (P < 0.05) and 8 +/- 7% (P = 0.31), respectively. 6. Extracts of purified human basophils liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by 17 +/- 3% (P < 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 +/- 1% (P < 0.0001) by a higher (5 microM) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 microM) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. 7. In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.     

Tumor necrosis factor-alpha-induced activation of RhoA in airway smooth muscle cells: role in the Ca2+ sensitization of myosin light chain20 phosphorylation

Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, has a potentially important role in the pathogenesis of bronchial asthma and may contribute to airway hyper-responsiveness. Recent evidence has revealed that TNF can increase the Ca(2+) sensitivity of agonist-stimulated myosin light chain(20) (MLC(20)) phosphorylation and contractility in guinea pig airway smooth muscle (ASM). In the present study, the potential intracellular pathways responsible for this TNF-induced Ca(2+) sensitization were investigated. In permeabilized cultured guinea pig ASM cells, recombinant human TNF stimulated an increase in Ca(2+)-activated MLC(20) phosphorylation under Ca(2+) "clamp" conditions. This increased MLC(20) phosphorylation was inhibited by preincubation with the Rho-kinase inhibitor Y27632. TNF also increased the proportion of GTP-bound RhoA, as measured using rhotekin Rho-binding domain, in a time course compatible with a role in the TNF-induced Ca(2+) sensitization. In cultured human ASM cells, recombinant human TNF also activated RhoA with a similar time course. In addition, TNF stimulated phosphorylation of the regulatory subunit of the myosin phosphatase, which was inhibited by Y27632. Although human ASM cells expressed both receptor subtypes, TNF-R1 and TNF-R2, the activation of RhoA was predominantly via stimulation of the TNF-R1, although RhoA did not immunoprecipitate with the TNF-R1. In conclusion, the TNF-induced increase in the Ca(2+) sensitivity of MLC(20) phosphorylation is through stimulation of the TNF-R1 receptor and via a RhoA/Rho-kinase pathway leading to inhibition of the myosin light chain phosphatase. This intracellular mechanism may contribute to TNF-induced airway hyper-responsiveness.     

Effects of nitroprusside, glyceryl trinitrate, and 8-bromo cyclic GMP on phosphorylase a formation and myosin light chain phosphorylation in rat aorta

The effects of nitroprusside (NP), glyceryl trinitrate (GTN), and the 8-bromo analog of cyclic GMP (8-Br-cGMP) on norepinephrine (NE)-stimulated phosphorylase a formation and myosin light chain (MLC) phosphorylation were examined in the rat aorta. NE produced a time-dependent increase in tension, phosphorylase a formation, and MLC phosphorylation. The formation of phosphorylase a and phosphorylation of MLC were transient, since both processes declined to basal levels within 30 min after the addition of NE even though tension remained elevated. NP and GTN inhibited tension, phosphorylase a formation, and MLC phosphorylation although inhibition of phosphorylase was greater when strips were treated with submaximal (i.e., 0.01 microM) NE concentrations. GTN was a more effective inhibitor of phosphorylase a formation than NP in NE-treated strips, although both agents and 8-Br-cGMP inhibited MLC phosphorylation. The guanylate cyclase inhibitor methylene blue (10 microM) effectively prevented the effects of NP and GTN. The results suggest that NP, GTN, and 8-Br-cGMP inhibit phosphorylase kinase and MLC kinase activation by lowering Ca2+ in the cell. This hypothesis is supported by the observations that 8-Br-cGMP inhibited the Ca2+-dependent, KCl-induced phosphorylase a formation most markedly at reduced concentrations of extra-cellular Ca2+. In addition, neither NP, GTN, nor 8-Br-cGMP inhibited phosphorylase a formation in forskolin-treated tissues, which occurred in response to cAMP-dependent phosphorylation of phosphorylase b kinase.