Search

Background: Phosphorylation state of dopamine- and cAMP-regulated phosphoprotein, molecular weight 32 kDa (DARPP32) is crucial to understand drug-mediated synaptic plasticity. In this study, mechanisms underlying repeated cocaine-stimulated phosphorylation of DARPP32 at threonine 75 (pDARPP32-Thr75) were determined by investigating the hypothesis that activation of protein kinases and phosphatases coupled to glutamate signaling is necessary for the regulation of pDARPP32-Thr75 after repeated cocaine administration., Methods: Intracaudate drug infusions into the rat dorsal striatum followed by Western immunoblot analysis were mainly performed to test this hypothesis., Results: The results demonstrated that 7 repeated daily intraperitoneal injections of cocaine (20mg/kg) upregulated the expression of pDARPP32-Thr75. Increases in the cytosolic Ca(2+) concentrations followed by Ca(2+)-dependent protein kinase activation through stimulation of Ca(2+) channels in striatal neurons were necessary for the phosphorylation. Activation of protein phosphatases further regulated the phosphorylation state by deactivating pDARPP32-Thr75 and upstream protein kinases., Conclusion: These findings suggest that activation of protein kinases and phosphatases coupled to glutamate receptors controls the phosphorylation state of DARPP32-Thr75 after repeated exposure to cocaine in the dorsal striatum in a Ca(2+)-dependent manner., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

Inhibitors of the sphingosine-1-phosphate (S1P) degrading enzyme S1P lyase (SPL) may be useful in the therapy of inflammatory diseases by preventing lymphocyte recruitment to diseased tissues. Here we describe a cellular assay for such inhibitors, which takes advantage of the observation that a fraction of the intracellular S1P accumulated in the presence of SPL inhibitors is secreted into the medium of cultured cells. The secreted S1P is then quantified using an S1P-sensitive reporter cell line. In the routine assay protocol, human HEK293T cells are treated with SPL inhibitors in the presence of phosphatase inhibitors and sphingosine; while the phosphatase inhibitors are included to prevent the degradation of S1P secreted from the cells, sphingosine is added as source for intracellular S1P that is prone to SPL degradation. The secreted S1P in the supernatant of the cell cultures is then quantified by measuring calcium flux induced in CHO-K1 cells expressing the human S1P3 receptor. Using this method SPL inhibitors were shown to induce a concentration-dependent increase of extracellular S1P under the conditions used; thus, the assay allows for the ranking of SPL inhibitors according to their potency on living cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Calyculin A (Caly A) is cell permeable toxin widely used in cell biology research as an inhibitor of type 1 and type 2A protein Ser/Thr phosphatases of the PPP family. Here we tested effects of low concentrations of Caly A on proliferation of human cancer and non-cancer cell lines. We found that long-term 0.3 nM Caly A prevented G1 to S phase cell cycle progression in human Hs-68 fibroblasts and ARPE19 epithelial cells, but not human breast cancer MDA-MB-468, MDA-MB-231 and MCF7 cells. These conditions produced no change in cyclin D1 levels or in the phosphorylation of endogenous proteins. However, acute application of 0.3 nM Caly A blocked serum-induced increase in intracellular calcium levels in Hs-68 fibroblasts, but not in MDA-MB-468 breast cancer cells. We propose that subnanomolar Caly A prevents cell cycle progression because it blocks calcium uptake by fibroblasts. This probably involves non-selective cation channels and cancer cell proliferation was not affected because calcium enters these cells by other channels. Our results suggest that calyculin A has dual actions and acts as a channel blocker, in addition to its well-established effects as a phosphatase inhibitor.

This report investigates acute changes in the sensitivity of 5-HT(1A) receptors in dorsal raphe (dr) neurons in response to elevated serotonin. DR neurons were isolated from adult rats and measurements of inhibition of Ca(2+) current by 5-HT were obtained using the whole cell patch clamp technique. During a 10-min application of 5-HT (with normal [Ca(2+)](i) approximately 100 nM) a desensitization occurred. The response to 20 nM 5-HT decreased by 66% relative to control and remained depressed for about 30 min. When the internal [Ca(2+)] was buffered to <1 nM only a weak transient desensitization occurred that was surmountable with higher [5-HT]. Adenylyl cyclase activation with forskolin mimicked the desensitization and selective inhibition of protein kinase A (PKA), but not protein kinase C (PKC), partially antagonized the desensitization induced by 5-HT. To measure the activity of PKA and phosphatase enzymes, dr slices were incubated with the selective agonist dipropyl-5-carboxamidotryptamine (DP-5-CT, 1 microM) for 10 min and the phosphorylation of the PKA substrate Kemptide was followed using ATP-gamma(32)P. DP-5-CT inhibited the cAMP stimulated maximal activity of PKA but raised basal PKA activity, thus increasing the percentage of PKA in the active state (activity ratio), an effect that was prevented by the selective 5-HT(1A) antagonist WAY100635. DP-5-CT also caused a significant inhibition of phosphatase activity. These data support a model in the dr where 5-HT(1A)-receptor stimulation of PKA promotes phosphorylation of a target and phosphatase inhibition leading to heterologous desensitization. The effect would be expected to have physiological consequences for 5-HT-mediated inhibitory post synaptic potentials and the Ca(2+) component of the action potentials of dr neurons., (Published by Elsevier Ltd.)

Using a combination of fluorescence measurements of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca(2+) entry stimulated by activation of the Ca(2+) release-activated Ca(2+) (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type 1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca(2+)](i) to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca(2+) rise associated with activation of CRAC uptake and inhibited Mn(2+) entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca(2+) stores is accompanied by a large depolarisation which is absolutely dependent upon Ca(2+) entry via the CRAC uptake pathway. Application of calyculin A or okadaic acid, a structurally unrelated phosphatase antagonist inhibits this depolarisation. Taken in concert, these data demonstrate a marked sensitivity of the CRAC entry pathway to inhibition by calyculin A and okadaic acid.

Excitotoxic cell death (ECD) is characteristic of mammalian brain following min of anoxia, but is not observed in the western painted turtle following days to months without oxygen. A key event in ECD is a massive increase in intracellular Ca(2+) by over-stimulation of N-methyl-d-aspartate receptors (NMDARs). The turtle's anoxia tolerance may involve the prevention of ECD by attenuating NMDAR-induced Ca(2+) influx. The goal of this study was to determine if protein phosphatases (PPs) and intracellular calcium mediate reductions in turtle cortical neuron whole-cell NMDAR currents during anoxia, thereby preventing ECD. Whole-cell NMDAR currents did not change during 80 min of normoxia, but decreased 56% during 40 min of anoxia. Okadaic acid and calyculin A, inhibitors of serine/threonine PP1 and PP2A, potentiated NMDAR currents during normoxia and prevented anoxia-mediated attenuation of NMDAR currents. Decreases in NMDAR activity during anoxia were also abolished by inclusion of the Ca(2+) chelator -- BAPTA and the calmodulin inhibitor -- calmidazolium. However, cypermethrin, an inhibitor of the Ca(2+)/calmodulin-dependent PP2B (calcineurin), abolished the anoxic decrease in NMDAR activity at 20, but not 40 min suggesting that this phosphatase might play an early role in attenuating NMDAR activity during anoxia. Our results show that PPs, Ca(2+) and calmodulin play an important role in decreasing NMDAR activity during anoxia in the turtle cortex. We offer a novel mechanism describing this attenuation in which PP1 and 2A dephosphorylate the NMDAR (NR1 subunit) followed by calmodulin binding, a subsequent dissociation of alpha-actinin-2 from the NR1 subunit, and a decrease in NMDAR activity.

In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion from the pancreatic beta-cell is an early pathogenetic event, but the mechanisms involved in glucose sensing are poorly understood. A messenger role has been postulated for L-glutamate in linking glucose stimulation to sustained insulin exocytosis in the beta-cell, but the precise nature by which L-glutamate controls insulin secretion remains elusive. Effects of L-glutamate on the activities of ser/thr protein phosphatases (PPase) and Ca(2+)-regulated insulin exocytosis in INS-1E cells were investigated. Glucose increases L-glutamate contents and promotes insulin secretion from INS-1E cells. L-glutamate also dose-dependently inhibits PPase enzyme activities analogous to the specific PPase inhibitor, okadaic acid. L-glutamate and okadaic acid directly and non-additively promote insulin exocytosis from permeabilized INS-1E cells in a Ca(2+)-independent manner. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by glucose-derived L-glutamate, may be a novel regulatory mechanism linking glucose sensing to sustained insulin exocytosis.

We previously demonstrated that histamine synthesis is drastically induced upon sensitization with an anti-DNP IgE clone, SPE-7, in IL-3-dependent mouse bone marrow derived mast cells (BMMC). We found that Ca2+ mobilization induced by SPE-7 exhibited a similar profile to the capacitative Ca2+ entry evoked by thapsigargin. Potentials for activation of mast cells were found to vary between different IgE clones, and a monovalent hapten, DNP-lysine, suppressed the activation induced by SPE-7. Ca2+ mobilization induced by SPE-7 was suppressed potently by the specific store-operated Ca2+ channel inhibitor, SK&F 96365, but not at all by Ca2+ channel inhibitors with more broad spectrum, La3+ and Gd3+, whereas the Ca2+ mobilization induced by Ag stimulation was suppressed by these inhibitors. Ca2+ mobilization was also induced by SPE-7 in in vitro differentiated mast cells, although the increases in histamine synthesis and IL-6 release were smaller than those in BMMC. These results suggest that Ca2+ influx operated by a distinct mechanism from that in Ag stimulation is essential for increased histamine synthesis and IL-6 release in mast cells.

Ca2+-sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC delta or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPgammaS elicited Ca2+-sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca2+-sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP.RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPgammaS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.

Ca-activated Cl currents (I(Cl(Ca))) are used frequently as reporters in functional studies of anesthetic effects on G protein-coupled receptors using Xenopus laevis oocytes. However, because anesthetics affect protein kinase C (PKC), they could indirectly affect I(Cl(Ca)) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1alpha and PP2A on I(Cl(Ca)) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca. X. laevis oocytes were studied under voltage clamp. Rat PP1alpha and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP(3)), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl(2) were directly injected into the oocyte. Responses to agonists (LPA 10(-6) M, IP(3) 10(-4) M, CaCl(2) 0.5 M) were measured at a holding potential of -70 mV in the presence or absence of the PP inhibitors cantharidin or OA. PP1 alpha and PP2A inhibited I(Cl(Ca)) from 7.6 +/- 0.9 microC to 2.5 +/- 0.9 microC and 3.2 +/- 1.4 microC, respectively. PP inhibition enhanced I(Cl(Ca)) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 alpha or PP2A. PKC inhibition by chelerythrine enhanced both LPA- and CaCl(2)-induced I(Cl(Ca)). Our data indicate that the Xenopus I(Cl(Ca)) is modulated by phosphorylation. This may complicate design and interpretation of studies of G protein-coupled receptors using this model.

The FAB mass spectrum of the Ca(2+) salt of RK-682 (1, MW 368), a potent protein tyrosine phosphatase inhibitor, shows a complex pattern due to Ca(2+) adduct ions with multimers of 1 and their decomposition ions. Addition of LiCl greatly simplified the FAB mass spectrum, providing a prominent Li(+) adduct ion of 1 at m/z 381 [M+2Li-H](+). The addition of LiCl also greatly simplified the FAB mass spectrum of calcium pantothenate. This approach may be generally useful for molecular weight determination of multivalent metal salts of organic compounds, or organic compounds that can form Li salts, by FAB mass spectrometry.

Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation-contraction coupling; furthermore abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca2+ sparks and SR Ca2+ load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca2+ sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca2+ stores, as determined by application of caffeine. These changes in Ca2+ release and SR Ca2+ load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-mediated RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR-mediated SR Ca2+ release leading to depleted SR Ca2+ stores in cardiac myocytes.

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).

Background: NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway., Methods and Results: The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+]i in depolarized RA (120 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of 10 micromol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 micromol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA-activating phospholipid sphingosine-1-phosphate (S1P, 12 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+]i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP-induced dilations., Conclusions: NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+-desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.

We showed that 5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium (GEA3162), a lipophilic nitric oxide (NO)-releasing agent, induced Ca(2+) entry into rat neutrophils in a concentration-dependent manner, whereas the guanylyl cyclase inhibitors, 6-anilino-5,8-quinolinequinone (LY83583) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), had no effect on GEA3162-induced response. The GEA3162-induced Ca(2+) entry was not observed in a Ca(2+)-free medium. GEA3162 did not potentiate but reduced the store-emptying activated Ca(2+) entry caused by cyclopiazonic acid. Stimulation of cells with GEA3162 in the absence of extracellular Ca(2+) followed by addition of cations showed that only Ca(2+) but not Ba(2+) and Sr(2+) entry occurs. Store-operated Ca(2+) entry was sensitive to La(3+) and Ni(2+) inhibition, whereas the GEA3162-induced Ca(2+) entry was sensitive to La(3+) but resistant to Ni(2+). cis-N-(2-Phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A) and calyculin A diminished the Ca(2+) entry activated by cyclopiazonic acid as well as by GEA3162. In contrast, 2-aminoethyldiphenyl borate (2-APB) diminished cyclopiazonic acid-but enhanced GEA3162-induced [Ca(2+)](i) change. Genistein effectively attenuated the cyclopiazonic acid-but slightly inhibited GEA3162-induced [Ca(2+)](i) change. Application of neomycin and high extracellular Ca(2+) concentration did not induce [Ca(2+)](i) rise. These data suggest that GEA3162 induced Ca(2+) entry and regulated Ca(2+) signal, through direct protein thiol oxidation. The action of GEA3162 demonstrates characteristics that distinguish it from the store-operated mechanism in neutrophils and therefore is likely to represent an entirely distinct pathway. Extracellular Ca(2+)-sensing receptor is not existing in neutrophils.

Smooth muscle contraction is primarily regulated by reversible phosphorylation of the 20-kDa light chains of myosin (MLC(20)) involving Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) and serine/threonine protein phosphatases (PP). The aim of this study was to investigate the effects of the protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) inhibitor cantharidin (Cant), its structural analogue endothall (ETA) and microcystin LR (MC) on force of contraction and MLC(20)-phosphorylation in arterial smooth muscle of mouse aorta. Cant increased force of contraction and MLC(20)-phosphorylation in intact arterial rings of mouse aorta in the presence of Ca(2+) whereas ETA and MC were ineffective under the same experimental conditions. In contrast, all compounds induced contraction and led to enhanced MLC(20)-phosphorylation in nominally Ca(2+)-free solution in fibers of mouse aorta permeabilised (skinned) with Triton X-100. In addition, Western blot analysis revealed that skinning of mouse aorta did not result in a loss of PP1 and PP2A compared to intact rings. Thus, both PP must be tightly bound to structural proteins, e.g. myosin. The findings indicate a Ca(2+)-independent mechanism of smooth muscle contraction involving inhibition of PP1- and/or PP2A-activities leading to enhanced force and MLC(20)-phosphorylation of arterial smooth muscle.

Calcium entry into mature erythrocytes (red blood cells; RBCs) is associated with multiple changes in cell properties. At low intracellular Ca(2+), efflux of potassium and water predominates, leading to changes in erythrocyte rheology. At higher Ca(2+) content, activation of kinases and phosphatases, rupture of membrane-to-skeleton bridges, stimulation of a phospholipid scramblase and phospholipase C, and induction of transglutaminase-mediated protein cross-linking are also observed. Because the physiologic relevance of these latter responses depends partially on whether Ca(2+) entry involves a regulated channel or nonspecific leak, we explored mechanisms that initiate controlled Ca(2+) influx. Protein kinase C (PKC) was considered a prime candidate for the pathway regulator, and phorbol-12 myristate-13 acetate (PMA), a stimulator of PKC, was examined for its influence on erythrocyte Ca(2+). PMA was found to stimulate a rapid, dose-dependent influx of calcium, as demonstrated by the increased fluorescence of an entrapped Ca(2+)-sensitive dye, Fluo-3/AM. The PMA-induced entry was inhibited by staurosporine and the PKC-selective inhibitor chelerythrine chloride, but was activated by the phosphatase inhibitors okadaic acid and calyculin A. The PMA-promoted calcium influx was also inhibited by omega-agatoxin-TK, a calcium channel blocker specific for Ca(v)2.1 channels. To confirm that a Ca(v)2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the alpha(1A) subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an omega-agatoxin-TK-sensitive, Ca(v)2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.

Alpha-1,4-Linked oligogalacturonides (OGs) are pectic fragments of the plant cell wall that are perceived by the plant cell as signalling molecules. Using cytosolic aequorin-expressing soybean (Glycine max L.) cells, we have analysed cytosolic Ca(2+) changes and the oxidative burst induced by OGs with different degrees of polymerization. Our results provide evidence that different OGs are sensed through transient elevations of cytosolic Ca(2+) that show different kinetics. Specificity of the Ca(2+) signature relies also on the precise structural characteristics of the OG molecules, such as the methylesterification of galacturonic acid residues and the steric conformation. Inhibition of the OG-induced Ca(2+) transient also blocks the oxidative burst, indicating that the cytosolic Ca(2+) increase is one of the earliest steps in OG-activated signalling. However, a phosphorylation event seems to precede the Ca(2+) rise, because the Ca(2+) transient could be abolished by the protein kinase inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). A pharmacological approach with different antagonists that interfere with the induction of the cytosolic Ca(2+) rise indicates that both extracellular Ca(2+) influx and intracellular Ca(2+) release participate in transducing the OG signal. Treatment of cells with OGs establishes a refractory state, which impairs the ability of the cell to respond to a second stimulus with the same elicitor for up to 16 h. This desensitization period could be prolonged with the phosphatase inhibitor okadaic acid, and eliminated with the protein kinase inhibitor Ro 31-8220, suggesting that phosphorylation events may be involved in the establishment of the cell refractory state.

Smooth muscle calcium sensitization reflects an inhibition of myosin light chain phosphatase (SMPP-1m) activity; however, the underlying mechanisms are not well understood. SMPP-1m activity can be modulated through phosphorylation of the myosin targeting subunit (MYPT1) by the endogenous myosin phosphatase-associated kinase, MYPT1 kinase (MacDonald, J. A., Borman, M. A., Muranyi, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2419-2424). Recombinant chicken gizzard MYPT1 (M130) was phosphorylated in vitro by a recombinant MYPT1 kinase, and the sites of phosphorylation were identified as Thr(654), Ser(808), and Thr(675). Introduction of recombinant MYPT1 kinase elicited a calcium-independent contraction in beta-escin-permeabilized rabbit ileal smooth muscle. Using an antibody that specifically recognizes MYPT1 phosphorylated at Thr(654) (M130 numbering), we determined that this calcium-independent contraction was correlated with an increase in MYPT1 phosphorylation. These results indicate that SMPP-1m phosphorylation by MYPT1 kinase is a mechanism of smooth muscle calcium sensitization.

The hydrolysis of cyclic nucleotide second messengers takes place through multiple cyclic nucleotide phosphodiesterases (PDEs). The significance of this diversification is not fully understood. Here we report the differential regulation of low K(m) Ca2+-activated (PDE1C) and Ca2+-independent, rolipram-sensitive (PDE4) PDEs by protein phosphorylation in the neuroendocrine cell line AtT20. Incubation of cells with 8-(4-chlorophenylthio)-cyclic AMP (CPT-cAMP) enhanced PDE4 and reduced PDE1C activity. These effects were blocked by H89 indicating mediation by cAMP-dependent protein kinase (PKA), furthermore in broken cell preparations PKA produced the same reciprocal changes of PDE activities. Calyculin A, an inhibitor of protein phosphatases 1 and 2 A, stimulated PDE4 and enhanced the inhibitory effect of CPT-cAMP on PDE1C. The reduction of PDE1C activity was characterized by a marked attenuation of the activation by Ca2+/calmodulin. Stimulation of PDE4 activity by CPT-cAMP or calyculin A was attributable to PDE4D3 and these effects could also be reproduced in human embryonic kidney cells expressing epitope-tagged PDE4D3. Together, these data show reciprocal regulation of PDE1C and PDE4D by PKA, which represents a novel scheme for plasticity in intracellular signalling.

Mechanical signals are important both as environmental and endogenous developmental cues in plants. Among the quickest measurable responses to mechanical stimulation (MS) in plants is the up-regulation of specific genes, including TCH3, in Arabidopsis. Little is known about the signaling events and components that link perception of mechanical signals to gene expression in plants. Calcium has been identified previously as being potentially involved, and a role for ethylene has also been suggested. Using the protein kinase inhibitor staurosporine, we determined that MS up-regulation of TCH3 expression requires protein kinase activity in young Arabidopsis seedlings. Our data from studies on the Arabidopsis ein6 mutant demonstrate that the EIN6 protein is also required, but that its role in mechanically induced TCH3 expression appears to be independent of ethylene. Challenge of seedlings with protein phosphatase inhibitors calyculin A and okadaic acid stimulated TCH3 expression even in the absence of MS, implying protein phosphatase activity acting to negatively regulate TCH3 gene expression. This phosphatase activity acts either downstream or independently of EIN6. EIN6 and protein kinase activity, on the other hand, operate downstream of calcium to mediate mechanically stimulated TCH3 expression.

The suppressive role of endogenous regucalcin (RC), which is a regulatory protein of calcium signaling, in the enhancement of protein phosphatase activity (PPA) in the cytosol and nucleus of kidney cortex in calcium-administered rats was investigated. Calcium content in the kidney cortex was significantly increased at 0.5-5 h after a single intraperitoneal administration of calcium chloride solution (10 mg Ca/100 g body weight) to rats. The analysis with Western blotting of RC protein showed that RC levels in the cytosol and nucleus were significantly increased 0.5-5 h after the administration of calcium (10 mg/100 g). PPA toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol and nucleus of kidney cortex. PPA toward three phosphoamino acids in the cytosol and nucleus was significantly increased by the administration of calcium (10 mg/100 g). The presence of anti-RC monoclonal antibody (25 ng/ml) in the enzyme reaction caused a significant increase in PPA toward phosphotyrosine, phosphoserine, and phosphothreonine in the cytosol and nucleus of kidney cortex in normal rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA toward three phosphoamino acids in the cytosol and nucleus was significantly enhanced in calcium-administered rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA in the cytosol and nucleus of normal rats and calcium-administered rats was completely abolished by the addition of RC (10(- 6) M) in the enzyme reaction mixture. The present study suggests that endogenous RC suppresses the enhancement of PPA in the cytosol and nucleus of kidney cortex in calcium-administered rats., (Copyright 2002 Wiley-Liss, Inc.)

Synapsins are major neuronal phosphoproteins involved in regulation of neurotransmitter release. Synapsins are well established targets for multiple protein kinases within the nerve terminal, yet little is known about dephosphorylation processes involved in regulation of synapsin function. Here, we observed a reciprocal relationship in the phosphorylation-dephosphorylation of the established phosphorylation sites on synapsin I. We demonstrate that, in vitro, phosphorylation sites 1, 2, and 3 of synapsin I (P-site 1 phosphorylated by cAMP-dependent protein kinase; P-sites 2 and 3 phosphorylated by Ca(2+)-calmodulin-dependent protein kinase II) were excellent substrates for protein phosphatase 2A, whereas P-sites 4, 5, and 6 (phosphorylated by mitogen-activated protein kinase) were efficiently dephosphorylated only by Ca(2+)-calmodulin-dependent protein phosphatase 2B-calcineurin. In isolated nerve terminals, rapid changes in synapsin I phosphorylation were observed after Ca(2+) entry, namely, a Ca(2+)-dependent phosphorylation of P-sites 1, 2, and 3 and a Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6. Inhibition of calcineurin activity by cyclosporin A resulted in a complete block of Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6 and correlated with a prominent increase in ionomycin-evoked glutamate release. These two opposing, rapid, Ca(2+)-dependent processes may play a crucial role in the modulation of synaptic vesicle trafficking within the presynaptic terminal.

Downregulation of the L-type Ca(2+) current (I(Ca)) is an important determinant of the electrical remodeling of diseased atria. Using a rat model of heart failure (HF) due to ischemic cardiopathy, we studied I(Ca) in isolated left atrial myocytes with the whole-cell patch-clamp technique and biochemical assays. I(Ca) density was markedly reduced (1.7+/-0.1 pA/pF) compared with sham-operated rats (S) (4.1+/-0.2 pA/pF), but its gating properties were unchanged. Calcium channel alpha(1C)-subunit quantities were not significantly different between S and HF. The beta-adrenergic agonist isoproterenol (1 micromol/L) had far greater stimulatory effects on I(Ca) in HF than in S (2.5- versus 1-fold), thereby suppressing the difference in current density. Dialyzing cells with 100 micromol/L cAMP or pretreating them with the phosphatase inhibitor okadaic acid also increased I(Ca) and suppressed the difference in density between S and HF. Intracellular cAMP content was reduced more in HF than in S. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine had a greater effect on I(Ca) in HF than in S (76.0+/-11.2% versus 15.8+/-21.2%), whereas the inhibitory effect of atrial natriuretic peptide on I(Ca) was more important in S than in HF (54.1+/-4.8% versus 24.3+/-8.8%). Cyclic GMP extruded from HF myocytes was enhanced compared with S (55.8+/-8.0 versus 6.2+/-4.0 pmol. mL(-1)). Thus, I(Ca) downregulation in atrial myocytes from rats with heart failure is caused by changes in basal cAMP-dependent regulation of the current and is associated with increased response to catecholamines.

We have reported that Trichomonas vaginalis, the causative agent of the most common, non-viral sexually transmitted disease, bound to cover slips coated with fibronectin (FN) (Crouch & Alderete, Microbiol 1999 145: 2835-43). In this study, we extend that observation by showing that FN binding is specific, and we present data on the requirements of FN binding by T. vaginalis. Immunofluorescence and immuno-gold labelling readily detected FN throughout the trichomonal surface. Parasites bound to(125)I-labelled FN in a time- and concentration-dependent fashion. In the absence of protease inhibitor, iodinated FN was released from the trichomonad surface. Unlabelled FN specifically competed for binding in a concentration-dependent fashion with the(125)I-labelled FN. Interestingly, the amount of FN bound by T. vaginalis organisms was dependent on iron. High-iron-grown trichomonads acquired lower numbers of molecules but with 10-fold higher affinity than low-iron-grown organisms. Further, we show that for iron-replete organisms, calcium (Ca(2+)) at physiological levels increased amounts of bound FN. The increase in binding was rapid, occurring within 5 min of Ca(2+)addition, and required de novo protein synthesis. Co-incubation of live parasites with Ca(2+)in the presence of FN was necessary to increase the amount of FN bound. Treatment of trichomonads with okadaic acid, but not other phosphatase inhibitors, resulted in a 50% decrease in binding of FN, regardless of the presence of Ca(2+), suggesting a role for phosphatase in FN association. These results indicate that depending on the iron status of T. vaginalis organisms in vivo, Ca(2+)may influence trichomonad recognition and binding to FN during host parasitism., (Copyright 2001 Academic Press.)

An increase in the intracellular Ca(2+) concentration controls a diverse range of cell functions, including gene expression, apoptosis, adhesion, motility, and proliferation. We have investigated Ca(2+) regulation of gene expression in rat aortic smooth muscle cells. We found that the expression of nuclear factor regulated by interleukin 3 (NFIL3)/adenovirus E4 promoter-binding protein (E4BP4)/basic region/leucine zipper (bZIP) type of a transcription factor that has a very important function in cell survival, was activated by thapsigargin (TG). This activation was inhibited by chelation of extra- or intracellular Ca(2+), suggesting that the induction by TG was dependent on the elevation of [Ca(2+)](i). Specific inhibition of calcineurin or calcium/calmodulin-dependent protein kinase (CaM kinase) by chemical means impaired the TG-induced NFIL3/E4BP4 expression. Expression of dominant negative forms of calcineurin or nuclear factor of activated T cells (NFAT) inhibited the induction of NFIL3/E4BP4 mRNA by TG. These results suggest that intracellular Ca(2+) plays a critical role in regulating gene expression of NFIL3/E4BP4 by calcineurin/NFAT and CaM kinase signaling in vascular smooth muscle cells.

These studies examine characteristics of the quiescent period (timelag) of the free cytosolic calcium ([Ca++]i) elevation that follows stimulation of human basophils through the IgE receptor. Previous studies established that the [Ca++]i timelag was sensitive to the rate of ligand binding, but little else is known about this response characteristic. The [Ca++]i timelag could be lengthened using antigenic stimulation that is rapid but only weakly induces secretion: tenfold differences in the "strength" of the stimulus, as assessed by histamine release, are associated with threefold differences in the timelag. Inhibiting p53/56lyn kinase with low concentrations of the specific inhibitor, PP1, lengthened the [Ca++]i timelag dramatically. PP1 was also found to delay the onset of syk phosphorylation and histamine release. Staurosporine and genistein, which are known to inhibit early tyrosine kinases, had, at best, only modest effects on the [Ca++]i timelag. Specific inhibitors of protein kinase C (PKC) had no effect on the [Ca++]i timelag, and direct activation of PKC with PMA had only very modest effects on the timelag. Contrary to expectations, basophils with the so-called nonreleasing phenotype demonstrated an IgE-mediated [Ca++]i response at the single-cell level. However, the length of [Ca++]i timelag in nonreleasing basophils was threefold longer than normally found in releasing basophils. Furthermore, the [Ca++]i response was significantly more asynchronous than in releasing basophils and lacking in a sustained [Ca++]i elevation. These studies indicate that the [Ca++]i timelag following stimulation through the IgE receptor is sensitive to inhibition of lyn kinase but not other agents that have been demonstrated to inhibit early tyrosine kinases previously. However, only one characteristic of the [Ca++]i response phenotype of nonreleasing basophils--the [Ca++]i timelag but not the absence of a sustained [Ca++]i elevation--could be mimicked by inhibition of lyn kinase with PP1.

RK-682 was reported to be a potent protein tyrosine phosphatase inhibitor. We found that (R)-3-hexadecanoyl-5-hydroxymethyltetronic acid (1) was easily converted to its calcium salt during column chromatography on Silica gel 60, and this calcium salt was identical to RK-682 originally isolated from a natural source. Here we report details of the asymmetric synthesis of (R)-1 and its conversion to the calcium salt. Fast atom bombardment mass spectrometric (FAB-MS) analysis of the free and calcium salt forms of RK-682 is also reported.

HSF-1 is regulated at multiple molecular levels through intra- and intermolecular protein-protein interactions as well as by post-translational modification through phosphorylation. We have found that elevating intracellular calcium ion levels by exposure to the ionophore A23187 or thapsigargin inhibits the conversion of HSF-1 from a latent cytoplasmic form to its nuclear/DNA binding form. To examine a role for calcium/calmodulin regulated enzymes in this process, we examined the ability of specific inhibitors to abrogate the effects of calcium elevation. While the inhibitor of calmodulin dependent kinase II, KCN62 enhanced activation of HSF-1 during heat shock, it failed to block the inhibitory effects of calcium increase. By contrast, the immunosuppresant drugs cyclosporin A and FK506 abolished the effects of calcium elevation on HSF-1 activation. As the biological effects of the drugs are effected through inhibition of the calcium/calmodulin regulated phosphatase calcineurin, this suggests a role for calcineurin in antagonizing HSF-1 activity. The experiments suggest the existence of phosphorylated residue(s) in HSF-1 important in one or more of the processes that lead to activation (trimerization, nuclear localization, DNA binding) and which becomes dephosphorylated due to the activation of a calcium/calmodulin/calcineurin complex.

The expression of Ca(2+)-binding protein regucalcin and its role in the regulation of protein phosphatase activity in rat brain neuronal cells obtained with primary culture was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in brain neuronal cells using rat regucalcin-specific primers. Moreover, regucalcin protein in brain neuronal cells was detected by Western blot analysis using a polyclonal rabbit anti-regucalcin antibody. The presence of anti-regucalcin monoclonal antibody (20 or 50 ng/ml) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward phosphotyrosine, phosphoserine and phosphothreonine in the reaction mixture containing the cytosol of neuronal cell homogenates. This increase was completely prevented by the addition of regucalcin (10(-8) M). Protein phosphatase activity toward three phosphoaminoacids was significantly elevated by the addition of Ca(2+) (100 microM) and calmodulin (5 microg/ml). This elevation was completely blocked by the addition of regucalcin (10(-8) M). The present study demonstrates that regucalcin is expressed in rat brain neuronal cells, and that it has an inhibitory effect on protein phosphatase activity in the cells.

1. Microcystin-LR, a specific and effective inhibitor of serine/threonine phosphatases type 1/2A which does not permeate cells, was used to distinguish intracellular and extracellular effects of phosphatase inhibitors on insulin secretion by RINm5F cells. 2. Incubation of intact RINm5F cells with microcystin-LR (0.1 - 2 microM) almost doubled basal insulin release at 3 mM glucose but left maximal insulin release induced by KCl (30 mM) unaffected. 3. In parallel, there was an increase in cytosolic Ca(2+) by up to half maximum, which could be suppressed by the Ca(2+)-channel blocker D600. 4. In contrast, microcystin-LR incubation of intact cells did not affect phosphatase activity but significantly reduced phosphatase activity when used in cellular fractions. 5. From these data we conclude that microcystin-LR could affect Ca(2+)-channels and insulin release by inhibiting an extracellular phosphatase-like activity.

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.

We have previously reported that sucrose modulates anthocyanin biosynthesis in cell suspension cultures of Vitis vinifera L. The main role of sugar in this response does not seem to be that of general carbohydrate source for the supply of energy. In the present work, a number of pharmacological agents were used to further investigate the components of the signal transduction pathway involved in the induction of anthocyanin biosynthesis by sugar. We found that the phosphorylation of hexose by hexokinase, but not its transport, has to be taken into account for the sucrose signal transduction leading to anthocyanin accumulation. Indeed, 3-O-methylglucose, a glucose analog transported into cells but not phosphorylated by hexokinase, has no effect on anthocyanin production. Mannose mimics the effect of sucrose in grape cells, and mannoheptulose, a specific inhibitor of hexokinase, reduces the accumulation of anthocyanins in response to sucrose. The results with the two latter analogs are discussed. Ca2+ channel blockers, verapamil and LaCl3, which were used to investigate the role of extracellular Ca2+, all inhibited the sugar response. Ca2+ depletion by pretreatment with ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) also blocked the sugar response, which was partially recovered when Ca2+ was added exogenously after Ca2+ depletion. The use of two potent calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphtalenesulphonamide (W7) and chlorpromazine, showed that calmodulin is involved in the sugar signal transduction. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), and the protein phosphatase inhibitors, endothall and cantharidin, also inhibited the sugar response. The results of the present study suggest the involvement of several components of general signal transduction pathways such as Ca2+, calmodulin, and protein kinases phosphatases in the induction of anthocyanin biosynthesis by sugar.

Purpose: To investigate the role of protein kinase C (PKC) in cholinergic agonist-induced Ca2+ elevation in lacrimal gland acini., Methods: Lacrimal gland acini were prepared by collagenase digestion, and changes in intracellular Ca2+ ([Ca2+]i) were measured using fura-2 as a fluorescent probe., Results: Preactivation of PKC by phorbol 12-myristate 13-acetate (PMA), or inhibition of protein phosphatase type 1/2A (PP1/2A) by calyculin A, decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by increasing concentrations of carbachol, a cholinergic agonist. Staurosporine, an inhibitor of PKC, completely reversed the effect of PMA. Inhibition of the Ca(2+)-independent PKC isoforms PKCdelta and -epsilon, but not the Ca(2+)-dependent isoform PKCalpha substantially reversed the inhibitory effect of PMA on cholinergic agonist-induced Ca2+ elevation. The inhibitory effect of PMA was obtained only in the presence of extracellular Ca2+, suggesting that PKC inhibits the influx of Ca2+. PMA completely inhibited the cholinergic agonist-induced plateau of [Ca2+]i. PMA and calyculin A decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by thapsigargin, further supporting the idea that PKC modulates the entry of Ca2+., Conclusions: In the lacrimal gland, agonist-induced changes in [Ca2+]i are negatively regulated by PKC-dependent phosphorylation of a target protein(s) that is sensitive to PP1/2A.

Ca2+ sensitization of smooth muscle contraction involves the small GTPase RhoA, inhibition of myosin light chain phosphatase (MLCP) and enhanced myosin regulatory light chain (LC20) phosphorylation. A potential effector of RhoA is Rho-associated kinase (ROK). The role of ROK in Ca2+ sensitization was investigated in guinea-pig ileum. Contraction of permeabilized muscle strips induced by GTPgammaS at pCa 6.5 was inhibited by the kinase inhibitors Y-27632, HA1077 and H-7 with IC50 values that correlated with the known Ki values for inhibition of ROK. GTPgammaS also increased LC20 phosphorylation and this was prevented by HA1077. Contraction and LC20 phosphorylation elicited at pCa 5.75 were, however, unaffected by HA1077. Pre-treatment of intact tissue strips with HA1077 abolished the tonic component of carbachol-induced contraction and the sustained elevation of LC20 phosphorylation, but had no effect on the transient or sustained increase in [Ca2+]i induced by carbachol. LC20 phosphorylation and contraction dynamics suggest that the ROK-mediated increase in LC20 phosphorylation is due to MLCP inhibition, not myosin light chain kinase activation. In the absence of Ca2+, GTPgammaS stimulated 35S incorporation from [35S]ATPgammaS into the myosin targeting subunit of MLCP (MYPT). The enhanced thiophosphorylation was inhibited by HA1077. No thiophosphorylation of LC20 was detected. These results indicate that ROK mediates agonist-induced increases in myosin phosphorylation and force by inhibiting MLCP activity through phosphorylation of MYPT. Under Ca2+-free conditions, ROK does not appear to phosphorylate LC20 in situ, in contrast to its ability to phosphorylate myosin in vitro. In particular, ROK activation is essential for the tonic phase of agonist-induced contraction.

We have reported that treatment with okadaic acid, a potent protein phosphatase inhibitor, has the ability to enhance the synthesis of the 78-kDa glucose-regulated protein (GRP78). This article reports our investigation of another protein phosphatase inhibitor, calyculin A, demonstrating the signaling pathways elicited by the protein phosphatase inhibitors that lead to the induction of grp78. Our data showed that the induction process is abolished by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Phosphorylation-activation of p38(MAPK) in the treated cells was indicated by its own phosphorylation, as shown by double Western blotting analyses and directly confirmed by the in vitro kinase assay using MAPK-activated protein kinase-2, a well-known downstream effector of p38(MAPK), as a substrate. The involvement of p38(MAPK) in this process is further substantiated by using transient transfection assays with a plasmid, pGRP78-Luc, which contains a 0.72-kbp stretch of the grp78 promoter. By exploiting the same transfection assay, we demonstrated that the up-regulation of the grp78 promoter by the protein phosphatase inhibitors is suppressed in the presence of the cytoplasmic calcium chelator bis(aminophenoxy)ethane N,N'-tetraacetic acid, the mitochondria calcium uniporter inhibitor ruthenium red as well as the antioxidants N-acetyl cysteine and pyrrolidinedithiocarbamate. Taken together, our results lead us to conclude that treatment with the protein phosphatase inhibitors would activate the signaling pathways involving p38(MAPK) and mitochondrial calcium-mediated oxidative stress and that these pathways must act in concert in order to confer the induction of grp78 by okadaic acid and calyculin A., (Copyright 2000 Wiley-Liss, Inc.)

Several lines of evidence indicate that transduction of the hypoxic stimulus at the carotid body involves an increase in cytosolic Ca2+ ([Ca2+]i) via activation of voltage-gated Ca2+ channels in the glomus cells. However, reported responses to hypoxia include either no effect on or inhibition of Ca2+ current in glomus cells. The apparent discrepancy between the effects of hypoxia on [Ca2+]i and Ca2+ channel activity prompted us to re-examine the effects of low oxygen on Ca2+ currents in glomus cells. Experiments were performed on freshly dissociated glomus cells from rabbit carotid bodies. Ca2+ channel activity was monitored using the whole-cell configuration of the patch clamp technique with Ba2+ as the charge carrier. Hypoxia (pO2 = 40 mmHg) augmented the Ca2+ current by 24% (at 0 mV). This augmentation was seen in a CO2/HCO3- but not in a HEPES buffered extracellular solution. However, when the extracellular pH (pHo) of a HEPES buffered solution is lowered from 7.4 to 7.0, then the Ca2+ current in glomus cells is augmented by hypoxia by 20%. Nisoldipine, an L-type Ca2+ channel blocker (2 microM), prevented augmentation of the Ca2+ current by hypoxia. On the other hand, an N- and P-type Ca2+ channel blocker (2 microM omega-conotoxin MVIIC) did not prevent the augmentation of the Ca2+ current by hypoxia. Protein kinase C (PKC) inhibitors, staurosporine (100 nM) and bisindolylmaleimide (2 microM), prevented augmentation by hypoxia. Okadaic acid (100 nM), an inhibitor of serine/threonine phosphatases also prevented augmentation of Ca2+ current by hypoxia; whereas, norokadaone, an inactive analog of okadaic acid, had no effect. These results suggest that hypoxia augments Ca2+ current through L-type Ca2+ channels via a PKC and/or phosphatase-sensitive pathways.

1 G protein-mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho-associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y-27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-induced contraction in alpha-toxin- or beta-escin-permeabilized rabbit trachea. 2 Although pre-treatment with Y-27632 dose-dependently inhibited GTPgammaS (10 microM)-induced Ca2+ sensitization of alpha-toxin-permeabilized trachea, a Y-27632-insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPgammaS response in the presence of Y-27632 (100 microM). 3 GF 109203X (5 microM) abolished 1 microM 4beta-phorbol 12, 13-dibutyrate (PDBu)-induced, but only partially inhibited the GTPgammaS-induced Ca2+ sensitization. A combination of Y-27632 (100 microM) and GF 109203X (5 microM) totally abolished the GTPgammaS response. 4 GTPgammaS caused only a small contraction in the absence of Ca2+. Wortmannin (30 microM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+-induced contraction. ATP-triggered contraction of the strip which had been treated with calyculin A (1 microM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 microM), but not by Y-27632 (100 microM), in the presence of GTPgammaS and Ca2+. 5 GTPgammaS, but not PDBu, contracted the beta-escin-permeabilized trachea in the absence of Ca2+, but the presence of Ca2+-independent MLCK. 6 We conclude that ROCK plays a primary role in G-protein-mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle.

The elusive coupling between endoplasmic reticulum (ER) Ca2+ stores and plasma membrane (PM) "store-operated" Ca2+ entry channels was probed through a novel combination of cytoskeletal modifications. Whereas coupling was unaffected by disassembly of the actin cytoskeleton, in situ redistribution of F-actin into a tight cortical layer subjacent to the PM displaced cortical ER and prevented coupling between ER and PM Ca2+ entry channels, while not affecting inositol 1,4,5-trisphosphate-mediated store release. Importantly, disassembly of the induced cortical actin layer allowed ER to regain access to the PM and reestablish coupling of Ca2+ entry channels to Ca2+ store depletion. Coupling is concluded to be mediated by a physical "secretion-like" mechanism involving close but reversible interactions between the ER and the PM.

C2-ceramide but not inhibitors of phosphatase types 1 and 2A (okadaic acid, calyculin A, tautomycin) blocked store-regulated Ca2+ entry induced in human neutrophils by thapsigargin. This contrasts with previous results showing that both types of compounds inhibit Ca2+ influx in fmet-leu-phe-treated cells. In present studies, phosphatase inhibitors increased the rate of secondary Ca2+ influx in a temperature-dependent manner. Their mechanism of action appeared to be independent of phosphatase inhibition since the inactive congeners, norokadaone and tetraacetyl okadaic acid, also potentiated Ca2+ influx at similar concentrations. When Ca2+ stores were predischarged by thapsigargin, okadaic acid but not norokadaone acted synergistically with fMLP to inhibit subsequent Ca2+ entry. Results suggest that blockade of Ca2+ influx in neutrophils is mediated by a phosphorylation reaction that is prolonged by phosphatase inhibitors. The requisite phosphorylation occurs in fMLP-activated cells but may be absent in cells incubated with thapsigargin.

The role of serine/threonine protein phosphatases PP1 and PP2A in mast cell secretion was investigated using the phosphatase inhibitors okadaic acid and calyculin A. Calyculin A (5-25 nm) inhibited antigen-induced secretion from a rat mucosal mast cell line (RBL-2H3) when added in conjunction with the activator. Okadaic acid (250-1000 nm) inhibited secretion only when added before activation and did so in a time- and concentration-dependent manner. Both inhibitors caused the cells to become rounder, but only calyculin A induced membrane blebbing and a loss of adherence. Okadaic acid also inhibited secretion induced by the calcium ionophore A23187, in the presence or absence of PMA, indicating that the phosphatase inhibitors act on a component of the secretory pathway downstream of calcium mobilization. Okadaic acid increased the phosphorylation of a number of proteins, as did an analogue methyl okadaate, which also inhibited secretion, but less effectively. Okadaic acid induced the phosphorylation of triton-insoluble proteins of 55, 18 and 16 kDa. The 55 kDa protein was identified as vimentin and okadaic acid induced its partial translocation to the triton-soluble fraction. Our data indicate that full secretory function in mucosal mast cells requires phosphatase activity., (Copyright 1998 Academic Press.)

The regulatory effect of regucalcin on Ca2+/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca2+ (100 microM) and calmodulin (0.30 microM). These increases were clearly inhibited by the addition of regucalcin (0.50-1.0 microM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 microg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 microM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 microM) in the presence of Ca2+ (1 and 10 microM). This increase was completely inhibited by the presence of regucalcin (0.12 microM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 microM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca2+/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin.

1. We have studied the regulation of whole-cell chloride current in T84 colonic epithelial cells by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1, 2-bisdeoxy-1,2-bisfluoro-Ins(3,4,5,6)P4 (i.e. F2-Ins(3,4,5,6)P4). 2. Calmodulin-dependent protein kinase II (CaMKII) increased chloride current 20-fold. This current (ICl,CaMK) continued for 7 +/- 1.2 min before its deactivation, or running down, by approximately 60 %. This run-down was prevented by okadaic acid, whereupon ICl,CaMK remained near its maximum value for >= 14.3 +/- 0.6 min. 3. F2-Ins(3, 4,5,6)P4 inhibited ICl,CaMK (IC50 = 100 microM) stereo-specifically, since its enantiomer, F2-Ins(1,4,5,6)P4 had no effect at >= 500 microM. Dose-response data (Hill coefficient = 1.3) showed that F2-Ins(3,4,5,6)P4 imitated only the non-co-operative phase of inhibition by Ins(3,4,5,6)P4, and not the co-operative phase. 4. Ins(3,4,5,6)P4 was prevented from blocking ICl,CaMK by okadaic acid (IC50 = 1.5 nM) and microcystin (IC50 = 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P4 to function. The IC50 values indicate that more than one species of phosphatase was required. One of these may be PP1, since F2-Ins(3,4,5,6)P4-dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.

In the present study it was investigated whether cisplatin-treated murine peritoneal macrophages produce oncostatin M (OSM) and what is the underlying mechanism. The culture supernatants of cisplatin-treated macrophages significantly inhibited the proliferation of OSM-sensitive cell line A375. Within 15 min of cisplatin treatment significant OSM was synthesized and secreted by macrophages. Inhibitors of serine/threonine and protein tyrosine phosphatases augmented cisplatin-induced OSM production of macrophages. The protein kinase C and protein tyrosine kinase inhibitors significantly inhibited OSM production of cisplatin-treated macrophages. The OSM production of cisplatin-treated macrophages was also inhibited in the presence of Ca2+ chelators, Ca2+ channel blocker and calmodulin/calmodulin-dependent kinase inhibitors. These data suggest that OSM production of cisplatin-treated macrophages is regulated by opposing actions of phosphatases and kinases. It is also suggested that OSM production of cisplatin-treated macrophages is dependent on Ca2+, calmodulin and calmodulin-dependent kinase.

The slow Ca2+-activated K+ current, sIAHP, underlying spike frequency adaptation, was recorded with the whole cell patch-clamp technique in CA1 pyramidal neurons in rat hippocampal slices. Inhibitors of serine/threonine protein phosphatases (microcystin, calyculin A, cantharidic acid) caused a gradual decrease of sIAHP amplitude, suggesting the presence of a basal phosphorylation-dephosphorylation turnover regulating sIAHP. Because selective calcineurin (PP-2B) inhibitors did not affect the amplitude of sIAHP, protein phosphatase 1 (PP-1) or 2A (PP-2A) are most likely involved in the basal regulation of this current. The ATP analogue, ATP-gamma-S, caused a gradual decrease in the sIAHP amplitude, supporting a role of protein phosphorylation in the basal modulation of sIAHP. When the protein kinase A (PKA) inhibitor adenosine-3', 5'-monophosphorothioate, Rp-isomer (Rp-cAMPS) was coapplied with the phosphatase inhibitor microcystin, it prevented the decrease in the sIAHP amplitude that was observed when microcystin alone was applied. Furthermore, inhibition of PKA by Rp-cAMPS led to an increase in the sIAHP amplitude. Finally, an adenylyl cyclase inhibitor (SQ22, 536) and adenosine 3',5'-cyclic monophosphate-specific type IV phosphodiesterase inhibitors (Ro 20-1724 and rolipram) led to an increase or a decrease in the sIAHP amplitude, respectively. These findings suggest that a balance between basally active PKA and a phosphatase (PP-1 or PP-2A) is responsible for the tonic modulation of sIAHP, resulting in a continuous modulation of excitability and firing properties of hippocampal pyramidal neurons.

Adrenal glucocorticoids exert powerful effects on cellular excitability in neuroendocrine cells and neurons, although the underlying mechanisms are poorly understood. In metabolically intact mouse anterior pituitary corticotrope (AtT20) cells glucocorticoid-induced proteins render large conductance calcium-activated potassium (BK) channels insensitive to inhibition by protein kinase A (PKA). In this study we have addressed whether this action of glucocorticoids is mediated via protein phosphatase activity at the level of single BK channels. In isolated inside-out patches from control AtT20 cells BK channels (125 pS) were inhibited by activation of closely associated PKA. Pretreatment (2 h) of cells with 1 microM dexamethasone before patch excision did not modify the intrinsic properties or expression levels of BK channel alpha-subunits in AtT20 cells. However, PKA-mediated inhibition of BK channel activity in isolated patches from steroid-treated cells was severely blunted. This effect of steroid was not observed using adenosine 5'-O-(3-thiotriphosphate) as phosphate donor or on exposure of the intracellular face of the patch with 10 nM of the protein phosphatase inhibitors okadaic acid or calyculin A but was mimicked by application of protein phosphatase 2A (PP2A) to the intracellular face of patches from control cells. Glucocorticoids did not modify total PP2A activity in AtT20 cells, suggesting that modified PP2A-like phosphatase activity closely associated with BK channels is required for glucocorticoid action.

Elevations in intracellular Ca2+ in electrically permeabilized islets of Langerhans produced rapid insulin secretory responses from beta-cells, but the Ca(2+)-induced secretion was not maintained and was irrespective of the pattern of administration of elevated Ca2+. Ca(2+)-insensitive beta-cells responded normally to activators of protein kinase C or cAMP-dependent kinase with increased insulin secretion. The loss of secretory responsiveness to Ca2+ was paralleled by a reduction in Ca(2+)-induced protein phosphorylation. This was caused by a reduction in Ca2+/calmodulin-dependent protein kinase II (CaMK II) activity in the desensitized cells, as assessed by measuring the phosphorylation of a CaMK II-specific exogenous substrate, autocamtide-2. The Ca(2+)-induced reductions in kinase activity and protein phosphorylation were not dependent on the activation of Ca(2+)-dependent protein kinases and were not caused by the activation of phosphoprotein phosphatases or of Ca(2+)-activated proteases. The concomitant reductions in CaMK II activity and Ca(2+)-induced insulin secretion suggest that the activation of CaMK II is required for normal insulin secretory responses to increased intracellular Ca2+ concentrations.

Mastoparan induced a transient elevation of cytosolic free Ca2+ concentration ([Ca2+]cyt) in tobacco suspension culture cells. The mastoparan-induced [Ca2+]cyt elevation was inhibited by 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl and neomycin but not by depletion of extracellular Ca2+, suggesting that the elevation was the result of Ca2+ release from the intracellular stores caused by stimulation of phosphoinositide turnover. Hydrogen peroxide which has been shown to induce an oxidative burst in soybean cells by mastoparan treatment [L. Legendre, P.F. Heinstein, P.S. Low, Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells, J. Biol. Chem., 267 (1992) 20140-20147], also induced a transient [Ca2+]cyt elevation in the tobacco cells. However, mastoparan did not induce an oxidative burst in the tobacco cells. Activation of a 50, a 75 and a 80 kDa protein kinases after the mastoparan-induced [Ca2+]cyt elevation was shown by an in-gel protein kinase assay. This activation was inhibited by neomycin, suggesting that the [Ca2+]cyt elevation is necessary for the mastoparan-induced activation of the protein kinases. The activation was inhibited also by pretreatment with staurosporine and was sustained by pretreatment with calyculin A, suggesting that the protein kinase activity is regulated by protein phosphorylation/dephosphorylation. The present report shows that mastoparan induces an increase in [Ca2+]cyt without oxidative burst and subsequent activation of protein kinases in tobacco cells.

1. Phosphorylation by kinases and dephosphorylation by phosphatases markedly affect the biological activity of proteins involved in stimulus-response coupling. In this study, we have characterized the effects of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, on insulin secretion. Mouse pancreatic islets were preincubated for 60 min in the presence of okadaic acid before their function was studied. 2. Okadaic acid dose-dependently (IC50 approximately 200 nM) inhibited insulin secretion induced by 15 mM glucose. At 0.5 microM, okadaic acid also inhibited insulin secretion induced by tolbutamide, ketoisocaproate and high K+, and its effects were not reversed by activation of protein kinases A or C. 3. The inhibition of insulin secretion did not result from an alteration of glucose metabolism (estimated by the fluorescence of endogenous pyridine nucleotides) or a lowering of the ATP/ADP ratio in the islets. 4. Okadaic acid treatment slightly inhibited voltage-dependent Ca2+ currents in beta cells (perforated patch technique), which diminished the rise in cytoplasmic Ca2+ (fura-2 method) that glucose and high K+ produce in islets. However, this decrease (25%), was insufficient to explain the corresponding inhibition of insulin secretion (90%). Moreover, mobilization of intracellular Ca2+ by acetylcholine was barely affected by okadaic acid, whereas the concomitant insulin response was decreased by 85%. 5. Calyculin A, another inhibitor of protein phosphatases 1 and 2A largely mimicked the effects of okadaic acid, whereas 1-norokadaone, an inactive analogue of okadaic acid on phosphatases, did not alter beta cell function. 6. In conclusion, okadaic acid inhibits insulin secretion by decreasing the magnitude of the Ca2+ signal in beta cells and its efficacy on exocytosis. The results suggest that, contrary to current concepts, both phosphorylation and dephosphorylation of certain beta cell proteins may be involved in the regulation of insulin secretion.

We have examined the electrophysiological responses induced by aluminium fluoride (AlF4-) and carbachol in Xenopus oocytes. Application of AlF4- induced Ca(2+)-dependent oscillatory and smooth Cl- currents. Pre-treatment of oocytes with microinjected guanosine 5'-O-(2-thiodiphosphate) diminished the currents, indicating that the effect of AlF4- occurred through G-protein activation. Confocal imaging of intracellular Ca2+ clearly demonstrated that AlF4- could increase the internal Ca2+ concentration in oocytes in the absence of external Ca2+. A protein kinase (PK) activator (4-beta-phorbol 12,13-dibutyrate) decreased the AlF4(-)-induced membrane currents, whereas a PK inhibitor (staurosporine) caused an increase. On the other hand, the protein phosphatase inhibitor (okadaic acid) showed little effect. Although the effects of the phosphorylating/dephosphorylating agents on the carbachol-induced currents were qualitatively similar to the case of AlF4-, some quantitative differences was noted. The results are discussed in terms of the signaling pathways involving muscarinic receptors and G-protein(s) in Xenopus oocytes.