Inhibitory Effect Of Wortmannin Research Articles

Intraoperative chlorhexidine-induced anaphylaxis suggesting an immunoglobulin-E-dependent mechanism indicated by basophil activation tests: two case reports

BackgroundAlthough chlorhexidine allergy has been shown to be mediated by immunoglobulin (Ig) E, few reports investigated the mechanism of chlorhexidine-induced anaphylaxis using basophil activation tests (BATs).Case presentationA 79-year-old man underwent cholecystectomy under general anesthesia. Anaphylaxis was diagnosed based on the clinical symptoms and high serum tryptase and histamine levels. Skin tests showed positive results only for chlorhexidine. Subsequently, BATs demonstrated that the causative agent was likely chlorhexidine. The inhibitory effect of wortmannin, an inhibitor of phosphoinositide 3-kinase, on basophil activation suggested an IgE-dependent mechanism underlying chlorhexidine-induced anaphylaxis.An 89-year-old man underwent inguinal hernioplasty under general anesthesia. Anaphylaxis was diagnosed based on the clinical symptoms and high serum tryptase and histamine levels. Skin tests and BATs with wortmannin were performed, showing similar results to case 1.ConclusionsBATs suggested an IgE-dependent mechanism for chlorhexidine-induced anaphylaxis and might be useful for investigating the mechanisms underlying drug-induced anaphylaxis.

Possible immunoglobulin-E-dependent sugammadex-induced anaphylaxis caused by an epitope other than \u03b3-cyclodextrin: a case report

BackgroundSugammadex is a synthetic γ-cyclodextrin derivative designed to selectively bind to steroidal neuromuscular blocking agents and reverse their effects. Although many cases of sugammadex-induced anaphylaxis have been reported, few studies have investigated the underlying mechanism.Case presentationA 55-year-old Japanese man underwent a laryngectomy under general anesthesia. One month before laryngectomy, he had undergone laryngoscopy under general anesthesia and received sugammadex administration without causing hypersensitivity. He had no history of allergies. The operation was finished without complications. Shortly after sugammadex administration, his blood pressure dropped to approximately 70 mmHg, and his heart rate increased to 110 beats/minute with systemic erythema. Suspecting anaphylaxis, he was treated with the intravenous injection of phenylephrine, d-chlorpheniramine, and hydrocortisone. After these treatments, his cardiovascular condition stabilized. Eight months after the event, skin prick tests and intradermal tests with all agents used during general anesthesia were performed. Intradermal tests showed positive results only for sugammadex. Subsequently, basophil activation tests with CD203c were performed using sugammadex, γ-cyclodextrin, and positive controls (anti-immunoglobulin-E and formyl-methionyl-leucyl-phenylalanine). In addition to both controls, sugammadex, but not γ-cyclodextrin, induced significant upregulation of CD203c expression. We performed additional basophil activation tests with wortmannin, an inhibitor of phosphoinositide 3-kinase, to investigate the mechanism underlying sugammadex-induced basophil activation. The inhibitory effect of wortmannin on basophil activation due to sugammadex was similar to that of anti-immunoglobulin-E, suggesting an immunoglobulin-E-dependent mechanism. Although the patient showed no hypersensitivity after the first exposure of sugammadex, anaphylaxis appeared after the second administration. Because most cases of sugammadex-induced anaphylaxis reportedly appeared after first administration, this seems to be a rare case.ConclusionsIn the present case, sugammadex-induced anaphylaxis might have occurred through an immunoglobulin-E-dependent mechanism and not involve γ-cyclodextrin as an epitope. Physicians should pay attention to the occurrence of sugammadex-induced anaphylaxis even when the patient has a history of safe administration of sugammadex.

Porphyromonas endodontalis Lipopolysaccharides Induce RANKL by Mouse Osteoblast in a Way Different from That of Escherichia coli Lipopolysaccharide

Introduction Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-κB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. Methods LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Toll-like receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. Results Both of the anti-TLR2 and anti-TLR4 antibodies significantly ( P < .05) inhibited the expression of RANKL from osteoblasts stimulated with P. endodontalis LPS; only anti-TLR2 antibody had a significant ( P < .05) inhibitory effect on E. coli LPS signaling. SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) prevented the up-regulation of RANKL expression in P. endodontalis LPS-infected osteoblasts ( P < .05). The inhibitory effect of wortmannin (phosphatidylinositol 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase-1/2 [MEK 1/2] inhibitor) were observed in E. coli LPS-treated mouse osteoblasts ( P < .05). Conclusions Results from this study showed that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4–JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin ( E. coli LPS).

Phosphatidylinositol 3-Kinase Facilitates Microtubule-dependent Membrane Transport for Neuronal Growth Cone Guidance

The activity of PI3K is necessary for polarized cell motility. To guide extending axons, environmental cues polarize the growth cone via asymmetric generation of Ca(2+) signals and subsequent intracellular mechanical events, including membrane trafficking and cytoskeletal reorganization. However, it remains unclear how PI3K is involved in such events for axon guidance. Here, we demonstrate that PI3K plays a permissive role in growth cone turning by facilitating microtubule (MT)-dependent membrane transport. Using embryonic chick dorsal root ganglion neurons in culture, attractive axon turning was induced by Ca(2+) elevations on one side of the growth cone by photolyzing caged Ca(2+) or caged inositol 1,4,5-trisphosphate. We show that PI3K activity was required downstream of Ca(2+) signals for growth cone turning. Attractive Ca(2+) signals, generated with caged Ca(2+) or caged inositol 1,4,5-trisphosphate, triggered asymmetric transport of membrane vesicles from the center to the periphery of growth cones in a MT-dependent manner. This centrifugal vesicle transport was abolished by PI3K inhibitors, suggesting that PI3K is involved in growth cone attraction at the level of membrane trafficking. Consistent with this observation, immunocytochemistry showed that PI3K inhibitors reduced MTs in the growth cone peripheral domain. Time-lapse imaging of EB1 on the plus-end of MTs revealed that MT advance into the growth cone peripheral domain was dependent on PI3K activity: inhibition of the PI3K signaling pathway attenuated MT advance, whereas exogenous phosphatidylinositol 3,4,5-trisphosphate, the product of PI3K-catalyzed reactions, promoted MT advance. This study demonstrates the importance of PI3K-dependent membrane trafficking in chemotactic cell migration.

Intracellular phenotype of Mycobacterium avium enters macrophages primarily by a macropinocytosis-like mechanism and survives in a compartment that differs from that with extracellular phenotype.

Mycobacterium avium uptake by human macrophages differs between the phenotypes of bacterium grown in laboratory media (extracellular growth, EG) and bacterium grown within macrophages (intracellular growth, IG). Studies in vivo have confirmed that, when spreading, pathogenic mycobacteria enter macrophages by a complement receptor 3-independent pathway, in contrast to mycobacteria uptake in vitro. M. avium, grown in macrophages (IG) for 3 or more days, invade fresh macrophages by a macropinocytosis-like mechanism, in contrast to bacteria grown in media (EG), confirmed by the inhibitory effect of wortmannin, an inhibitor of phosphoinoside-3-kinase, on the uptake of IG, but not EG, by macrophages. The IG phenotype was seen in vacuoles with lower pH than those inhabited by the EG phenotype. Incubation of macrophages with bafilomycin A1, an inhibitor of vacuole acidification, decreased the viability of intracellular IG, but not EG, phenotype, suggesting the importance of an acidic environment for the regulation of IG genes. In addition, the percentage of vacuoles that incorporate and retain LAMP-1 is smaller with EG than with IG bacteria. The formation of M. avium macropinosomes was also shown to be independent of microtubules. These data suggest that uptake of extracellular fluid is part of M. avium IG phenotype uptake by macrophages, and that the IG phenotype inhabits a slightly different vacuole than that of EG.

Sustained Induction of ERK, Protein Kinase B, and p70 S6 Kinase Regulates Cell Spreading and Formation of F-actin Microspikes Upon Ligation of Integrins by Galectin-8, a Mammalian Lectin

Galectin-8, a member of the galectin family of mammalian lectins, is a secreted protein that promotes cell adhesion and migration upon binding to a subset of integrins through sugar-protein interactions. Ligation of integrins by galectin-8 triggers a distinct pattern of cytoskeletal organization, including formation of F-actin-containing microspikes. This is associated with activation of integrin-mediated signaling cascades (ERK and phosphatidylinositol 3 kinase (PI3K)) that are much more robust and are of longer duration than those induced upon cell adhesion to fibronectin. Indeed, formation of microspikes is enhanced 40% in cells that overexpress protein kinase B, the downstream effector of PI3K. Inhibition of PI3K activity induced by wortmannin partially inhibits cell adhesion and spreading while largely inhibiting microspike formation in cells adherent to galectin-8. Furthermore, the inhibitory effects of wortmannin are markedly accentuated in cells overexpressing PKB or p70S6K (CHO(PKB) and CHO(p70S6K) cells), whose adhesion and spreading on galectin-8 (but not on fibronectin) is inhibited approximately 25-35% in the presence of wortmannin. The above results suggest that galectin-8 is an extracellular matrix protein that triggers a unique repertoire of integrin-mediated signals, which leads to a distinctive cytoskeletal organization and microspike formation. They further suggest that downstream effectors of PI3K, including PKB and p70 S6 kinase, in part mediate cell adhesion, spreading, and microspike formation induced by galectin-8.

A Selective Role for Phosphatidylinositol 3,4,5-Trisphosphate in the Gi-dependent Activation of Platelet Rap1B

The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G(i)-dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fcgamma receptor IIA (FcgammaRIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G(i)-dependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of FcgammaRIIA, by stimulation of the P2Y(12) receptor for ADP, or by epinephrine. However, in platelets from PI3Kgamma-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3Kgamma did not prevent the ability of epinephrine to potentiate platelet aggregation through a G(i)-dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), but not PtdIns(3,4)P(2), although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P(3) was able to relieve the inhibitory effect of apyrase on FcgammaRIIA-mediated platelet aggregation. We conclude that stimulation of a G(i)-dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P(3), but not PtdIns(3,4)P(2), and that this process may contribute to potentiation of platelet aggregation.

Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase–dependent pathway in rats

Background & Aims: We tested the hypothesis that during bile duct obstruction, increased biliary bile acids trigger cholangiocyte proliferation and secretion by a phosphatidylinositol 3-kinase (PI3-K)–dependent pathway. Methods: In bile duct–incannulated (BDI) rats, bile duct obstruction present for 7 days was relieved for 24 hours by external bile drainage. During the 24-hour drainage period, animals received either Krebs Ringer Henseleit (the bile-depleted group), or sodium taurocholate (the bile-depleted, taurocholate-infused group). We evaluated cholangiocyte proliferation and secretin-stimulated ductal secretion. Apical bile acid transporter (ABAT) expression and bile acid transport activity was determined. In pure preparations of cholangiocytes, we examined the effect of taurocholate (in the absence or presence of wortmannin or PI 3,4-bisphosphate the lipid product of PI3-K) on cholangiocyte proliferation and secretin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels. Results: Bile depletion reduced cholangiocyte proliferation and secretin-stimulated ductal secretion and ABAT expression and bile acid transport activity compared with 1-week BDI control rats. In bile-depleted, taurocholate-infused rats, cholangiocyte proliferation and secretion and ABAT expression and bile acid transport activity were maintained at levels similar to those seen in BDI control rats. In vitro, taurocholate stimulation of DNA replication and secretin-stimulated cAMP levels was blocked by wortmannin. The inhibitory effect of wortmannin on taurocholate stimulation of cholangiocyte proliferation and secretion was prevented by PI 3,4-bisphosphate. Conclusions: Bile acid uptake by ABAT and the PI3-K pathway are important for bile acids to signal cholangiocyte proliferation. In bile duct obstruction, increased biliary bile acid concentration and ABAT expression initiate increased cholangiocyte proliferation and secretion.GASTROENTEROLOGY 2002;123:1226-1237

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C.

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.

Wortmannin inhibits spreading and chemotaxis of rat osteoclasts in vitro.

Wortmannin (WT) and 17beta-hydroxywortmannin (HWT), which are inhibitors of phosphatidylinositol-3(OH)-kinase (PI3K), have been shown previously to inhibit bone resorption in vitro and in vivo, possibly by interfering with formation of the osteoclast ruffled border. Since migration of osteoclasts also plays an important role in the process of bone resorption, we investigated the effects of these inhibitors on osteoclast morphology and motility. Both HWT and WT caused a sustained decrease in the planar area of osteoclasts in vitro (half maximal effect at 25 and 165 nM, respectively), with the effect of HWT on cell area more readily reversible than WT. These agents also caused accumulation of intracellular vesicles. Time-lapse video microscopy was used to record the migration of osteoclasts in response to macrophage colony-stimulating factor (M-CSF) or vehicle, flowing passively from a micropipette positioned 200-400 microm from the cell. M-CSF caused directed migration of osteoclasts, indicating chemotaxis (over 3 h osteoclasts migrated 96 +/- 14 microm in response to M-CSF vs. 11 +/- 2 microm in control experiments). Both WT (100 or 500 nM) and LY294002 (100 microM), a specific PI3K inhibitor structurally unrelated to WT, significantly inhibited osteoclast chemotaxis in response to M-CSF. Taken together, these effects of WT, HWT, and LY294002 are consistent with an important role for PI3K in regulating cytoskeletal function in osteoclasts. The inhibitory effects of WT and HWT on bone resorption may be due, in part, to impairment of osteoclast motility.

Wortmannin inhibits both insulin- and contraction-stimulated glucose uptake and transport in rat skeletal muscle.

The role of phosphatidylinositol (PI) 3-kinase for insulin- and contraction-stimulated muscle glucose transport was investigated in rat skeletal muscle perfused with a cell-free perfusate. The insulin receptor substrate-1-associated PI 3-kinase activity was increased sixfold upon insulin stimulation but was unaffected by contractions. In addition, the insulin-stimulated PI 3-kinase activity and muscle glucose uptake and transport in individual muscles were dose-dependently inhibited by wortmannin with one-half maximal inhibition values of approximately 10 nM and total inhibition at 1 microM. This concentration of wortmannin also decreased the contraction-stimulated glucose transport and uptake by approximately 30-70% without confounding effects on contractility or on muscle ATP and phosphocreatine concentrations. At higher concentrations (3 and 10 microM), wortmannin completely blocked the contraction-stimulated glucose uptake but also decreased the contractility. In conclusion, inhibition of PI 3-kinase with wortmannin in skeletal muscle coincides with inhibition of insulin-stimulated glucose uptake and transport. Furthermore, in contrast to recent findings in incubated muscle, wortmannin also inhibited contraction-stimulated glucose uptake and transport. The inhibitory effect of wortmannin on contraction-stimulated glucose uptake may be independent of PI 3-kinase activity or due to inhibition of a subfraction of PI 3-kinase with low sensitivity to wortmannin.

Inhibitory effect of wortmannin on phosphatidylinositol 3-kinase-mediated cellular events

Inhibitory effect of wortmannin on phosphatidylinositol 3-kinase-mediated cellular events

Phosphatidylinositol 3‐kinase activity is not essential for CD28 costimulatory activity in Jurkat T cells: studies with a selective inhibitor, wortmannin

The interaction of CD28 with its counter-receptor, B7-1 (CD 80), on antigen-presenting cells induces a co-signal in T cells required to promote antigen-dependent interleukin-2 (IL-2) production and to prevent clonal anergy. CD28 stimulation causes both protein-tyrosine kinase and phosphatidylinositol3-kinase (PI3-K) activation, suggesting a possible role for these enzyme activities in CD28 co-signal transduction. Here, we investigate the effect of wortmannin, a selective and irreversible PI3-K inhibitor on CD28 co-signaling events in the Jurkat T cell line. Wortmannin added to cell cultures partially inhibits CD28-induced tyrosine phosphorylation of the putative p110 catalytic subunit of PI3-K, but does not block CD28-induced association of the p85 PI3-K regulatory subunit with the CD28 receptor. Wortmannin inhibits in a dose-dependent manner both total cellular PI3-K activity and CD28-induced receptor-associated PI3-K activity. Wortmannin (1 microM) inhibits cellular PI3-K activity by 90% with complete inhibition achieved at 10 microM. The inhibitory effect of wortmannin on cellular PI3-K activity is prolonged ( > 18 h), suggesting that the drug is not readily metabolized by Jurkat T cells. Wortmannin, at concentrations that blocked PI3-K activity, fails to inhibit the synergistic effect of CD28 on IL-2 secretion in the presence of phorbol 12-myristate 13-acetate and ionomycin. These data demonstrate that CD28-induced signaling events other than the activation of PI3-K catalytic activity contribute to the control of IL-2 secretion.

Wortmannin inhibits carcinogen-stimulated phosphorylation of ethanolamine and choline

We have previously reported that in C3H/10T1/2 fibroblasts the environmental carcinogen 7,12-dimethyl-benz[a]anthracene (DMBA) stimulated phosphorylation of ethanolamine (Etn). Here we show that in these fibroblasts DMBA also stimulates phosphorylation of choline (Cho). Wortmannin (50–200 nM), an established inhibitor of phosphatidylinositol-3-kinase (PI3K), significantly inhibited DMBA-induced phosphorylation of both Etn and Cho. Wortmannin also inhibited the effect of insulin, a major activator of PI3K, on DNA synthesis. However, insulin had no effect on the phosphorylation of Etn and Cho. These data suggest that a carcinogen-induced kinase phosphorylates both Etn and Cho, and that the inhibitory effect of wortmannin on Etn/Cho kinase activity may be unrelated to its inhibitory effect on PI3K activity.

Essential role of myosin light chain kinase in the mechanism for MgATP- dependent priming of exocytosis in adrenal chromaffin cells

Ca(2+)-induced exocytosis in chromaffin cells now seems to consist of at least two distinct steps:MgATP-dependent Ca(2+)-dependent priming of the secretory apparatus, and Ca(2+)-dependent MgATP-independent step that triggers exocytosis (Bittner and Holz, 1992). Recently we found that a specific inhibitor of myosin light chain kinase (MLCK), wortmannin, inhibits Ca(2+)-induced catecholamine release from digitonin-permeabilized chromaffin cells, suggesting an implication of MLCK in the mechanisms of Ca(2+)-induced exocytosis (Imaizumi et al., 1992b). To elucidate further the implication of MLCK in the mechanism of exocytosis, we studied the effects of wortmannin and a peptide inhibitor (SM-1) corresponding to the pseudosubstrate domain of MLCK on MgATP-dependent and MgATP-independent release in digitonin-permeabilized chromaffin cells. Ca(2+)-induced exocytosis from the permeabilized cells in the presence of MgATP was inhibited by both SM-1 and wortmannin. Inhibitory effect of wortmannin on the rate of release induced by 10 microM Ca2+ in the presence of MgATP was much prominent in the later phase (1-10 min), although the initial rate was also decreased. SM-1 strongly inhibited ATP-dependent release without affecting Ca(2+)-dependent ATP-independent release at all. In addition, priming effect of MgATP that underlies Ca(2+)-dependent ATP-independent release was remarkably reduced by both wortmannin and SM-1. These results suggest that MLCK plays an essential role in ATP-dependent priming of Ca(2+)-induced exocytosis in chromaffin cells.

Inhibition of agonist-stimulated inositol 1,4,5-trisphosphate production and calcium signaling by the myosin light chain kinase inhibitor, wortmannin.

The myosin light chain kinase inhibitor, wortmannin (WT), caused dose-dependent inhibition of the sustained increases but not the initial spikes of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and cytoplasmic Ca2+ concentration ([Ca2+]i) in angiotensin II (AII)-stimulated adrenal glomerulosa cells. In contrast, WT did not affect the [Ca2+]i increases evoked by K(+)-induced depolarization or by depletion of intracellular Ca2+ stores with ionomycin or thapsigargin, suggesting that its inhibitory effects on Ins-1,4,5-P3 and [Ca2+]i responses are closely related. The inhibitory effect of WT on the Ins-1,4,5-P3 response was not secondary to its reduction of Ca2+ entry, since ionomycin-induced elevation of [Ca2+]i did not restore Ins-1,4,5-P3 formation. Also, WT inhibited agonist- and GTP gamma S-stimulated Ins-1,4,5-P3 formation in permeabilized cells maintained at fixed Ca2+ concentrations. Thus, inhibition of Ca2+ influx by WT is a consequence of reduced Ins-1,4,5-P3 formation and could reflect the resultant refilling of the agonist-sensitive Ca2+ pool. Indeed, AII-depleted Ca2+ pools were found to refill by a thapsigargin-sensitive mechanism in WT-treated cells. Other structurally unrelated inhibitors of myosin light chain kinase (MS-347a, KT5926, and ML-9) also inhibited AII-induced inositol phosphate formation, albeit with lower potencies than WT. MS-347a, like WT, inhibited only the second phase of the inositol phosphate and [Ca2+]i responses to AII. These findings demonstrate that WT is a potent inhibitor of the Ins-1,4,5-P3-Ca2+ signaling pathway and acts primarily on a mechanism that promotes Ins-1,4,5-P3 formation and is sensitive to inhibitors of myosin light chain kinase activity.

P-151 - inhibitory effects of wortmannin on the release of neurotransmitters from isolated spinal cord of the newborn rat.

P-151 - inhibitory effects of wortmannin on the release of neurotransmitters from isolated spinal cord of the newborn rat.

Effects of wortmannin on alpha-1/alpha-2 adrenergic receptor-mediated contractile responses in rabbit vascular tissues.

The inhibitory effect of wortmannin (WO), a fungus-derived protein kinase inhibitor, was assessed on contractile responses elicited by phenylephrine-induced alpha 1-(alpha 1 R) and UK 14304-induced alpha 2-adrenergic receptor (alpha 2R) stimulation in the rabbit aorta and saphenous vein, respectively. In agonist dose-response studies, WO caused a noncompetitive inhibition of both alpha 1R and alpha 2R responses, but was more potent against alpha 2R. Maximally effective single-dose responses at both receptors were less sensitive to WO. The initial alpha 1R contractile response, associated with intracellular Ca2+ release and myosin light chain kinase activation, was relatively insensitive to WO, while the Ca2+ influx-dependent tonic contraction was more sensitive. Contractions induced by high K+ buffer were relatively insensitive to WO in both the aorta and saphenous vein. These results indicate that WO inhibits receptor-initiated Ca2+ influx-dependent contractile responses such as those caused by alpha 2R stimulation and the sustained phase of alpha 1R stimulation more readily than Ca2+ release-dependent responses.