Cytosolic Protein Kinase Activity Research Articles

FRET biosensors reveal AKAP‐mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

A‐Kinase Anchoring Proteins (AKAPs) serve as scaffolds for cAMP‐dependent Protein Kinase (PKA) and other signaling enzymes within specific subcellular compartments. However, some AKAPs are not static anchors, but form dynamic signaling complexes that can traffic to different subcellular compartments when triggered. Gravin (AKAP12) is a multivalent AKAP that anchors PKA and other enzymes to the plasma membrane but redistributes to the cytosol upon intracellular Ca2+ elevation. Through the redistribution of gravin, we postulate that Ca2+ may regulate the interaction of PKA with downstream substrates, thus representing a novel crosstalk mechanism between Ca2+ and PKA‐dependent pathways. To assess this, we measured the impact of gravin‐V5/His expression on compartmentalized PKA activity using the PKA FRET biosensor AKAR3. In cultured AN3 CA cells, which lack endogenous gravin, expression of gravin‐V5/His increased forskolin‐stimulated PKA activity in AKAR3 constructs targeted to the plasma membrane compared to control cells lacking gravin or expressing a ΔPKA‐gravin construct missing the PKA‐binding domain. In contrast, gravin‐V5/His expression decreased forskolin‐stimulated PKA activity in cytosolic AKAR3 constructs. A mutant gravin construct with inhibited membrane localization had no effect on membrane and cytosolic PKA activity. Finally, pretreatment with the calcium‐elevating agent thapsigargin caused the redistribution of gravin and prevented the gravin‐mediated increase in plasma membrane PKA activity following forskolin or isoproterenol treatment. These results reveal that Ca2+ alters receptor‐mediated PKA activity through gravin redistribution and supports the hypothesis that gravin mediates crosstalk between Ca2+ and PKA‐dependent pathways.

Spatiotemporally Regulated Protein Kinase A Activity Is a Critical Regulator of Growth Factor-Stimulated Extracellular Signal-Regulated Kinase Signaling in PC12 Cells

PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell proliferation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation. While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma membrane, although they generate temporally distinct activity patterns. We further show that both stimuli fail to induce cytosolic PKA activity and identify phosphodiesterase 3 (PDE3) as a critical regulator in maintaining this spatial compartmentalization. Importantly, inhibition of PDE3, and thus perturbation of the spatiotemporal regulation of PKA activity, dramatically increases the duration of EGF-stimulated nuclear ERK activity in a PKA-dependent manner. Together, these findings identify EGF and NGF as potent activators of PKA activity specifically at the plasma membrane and reveal a novel regulatory mechanism contributing to the growth factor signaling specificity achieved by NGF and EGF in PC12 cells.

Possible involvement of protein kinase c inhibition in the reduction of phorbol ester-induced neutrophil aggregation by magnolol in the rat.

The influence of the plant product magnolol on neutrophil aggregation has been investigated in the rat. Magnolol inhibited phorbol 12-myristate 13-acetate (PMA)-activated rat neutrophil aggregation in a concentration-dependent manner with an IC50 (concentration resulting in 50% inhibition) of 24.2 +/- 1.7 microM. Magnolol suppressed the enzyme activity of neutrophil cytosolic and rat brain protein kinase C (PKC) over the same range of concentrations at which it inhibited the aggregation. Magnolol did not affect PMA-induced cytosolic PKC-alpha and -delta membrane translocation or trypsin-treated rat-brain PKC activity, but attenuated [3H]phorbol 12,13-dibutyrate binding to neutrophil cytosolic PKC. These results suggest that the inhibition of PMA-induced rat neutrophil aggregation by magnolol is probably attributable, at least in part, to the direct suppression of PKC activity through blockade of the regulatory region of PKC.

Inhibition of the mitochondrial permeability transition by protein kinase A in rat liver mitochondria and hepatocytes

NO and cGMP administered at reperfusion after ischaemia prevent injury to hepatocytes mediated by the MPT (mitochondrial permeability transition). To characterize further the mechanism of protection, the ability of hepatic cytosol in combination with cyclic nucleotides to delay onset of the calcium-induced MPT was evaluated in isolated rat liver mitochondria. Liver cytosol plus cGMP or cAMP dose-dependently inhibited the MPT, required ATP hydrolysis for inhibition and did not inhibit mitochondrial calcium uptake. Specific peptide inhibitors for PKA (protein kinase A), but not PKG (protein kinase G), abolished cytosol-induced inhibition of MPT onset. Activity assays showed a cGMP- and cAMP-stimulated protein kinase activity in liver cytosol that was completely inhibited by PKI, a PKA peptide inhibitor. Size-exclusion chromatography of liver cytosol produced a single peak of cGMP/cAMP-stimulated kinase activity with an estimated protein size of 180-220kDa. This fraction was PKI-sensitive and delayed onset of the MPT. Incubation of active catalytic PKA subunit directly with mitochondria in the absence of cytosol and cyclic nucleotide also delayed MPT onset, and incubation with purified outer membranes led to phosphorylation of a major 31kDa band. After ischaemia, administration at reperfusion of membrane-permeant cAMPs and cAMP-mobilizing glucagon prevented reperfusion injury to hepatocytes. In conclusion, PKA in liver cytosol activated by cGMP or cAMP acts directly on mitochondria to delay onset of the MPT and protect hepatocytes from cell death after ischaemia/reperfusion.

PKA Activity Compartmentation Requires Slow Nuclear Transport Kinetics in Cardiac Myocytes

Segregated cytosolic and nuclear cAMP-dependent protein kinase (PKA) activity is important for specifying contractile vs. transcriptional phenotypes in cardiac health and disease. However, the molecular mechanisms selecting for activity in these different compartments remain uncertain. Here, we develop a biochemically mechanistic computational model of cytosolic and nuclear PKA activity to test competing hypotheses for regulating important cytosolic (PLB) and nuclear (CREB) PKA substrates in cardiac myocytes. Our model predicts a sustained 30 min exposure to isoproterenol fully activates PKA in both the cytosol and nucleus, while a transient 2 min exposure fully activates PKA in the cytosol but only achieves 44% max activation in the nucleus. Comparing experimentally measured (28 min) vs. hypothetically fast (2.8 min) PKA diffusion time constants, we show that nuclear PKA activation is rate-limited by translocation of PKA catalytic subunit (but not catalytic enzyme kinetics). In contrast, by blocking PKI-mediated nuclear export, we show that nuclear PKA activity termination is primarily determined by PKA deactivation (and not active nuclear transport). These simulated responses provide quantitative support for the hypothesis that slow nuclear transport kinetics are required for segregated PKA signaling in the cytosol and nucleus.

Ceramide regulation of nuclear protein import

Nucleocytoplasmic trafficking is an essential and responsive cellular mechanism that directly affects cell growth and proliferation, and its potential to address metabolic challenge is incompletely defined. Ceramide is an antiproliferative sphingolipid found within vascular smooth muscle cells in atherosclerotic plaques, but its mechanism of action remains unclear. The hypothesis that ceramide inhibits cell growth through nuclear transport regulation was tested. In smooth muscle cells, exogenously supplemented ceramide inhibited classical nuclear protein import that involved the activation of cytosolic p38 mitogen-activated protein kinase (MAPK). After application of SB 202190, a specific and potent pharmacological antagonist of p38 MAPK, sphingolipid impingement on nuclear transport was corrected. Distribution pattern assessments of two essential nuclear transport proteins, importin-alpha and Cellular Apoptosis Susceptibility, revealed ceramide-mediated relocalization that was reversed upon the addition of SB 202190. Furthermore, cell counts, nuclear cyclin A, and proliferating cell nuclear antigen expression, markers of cellular proliferation, were diminished after ceramide treatment and effectively rescued by the addition of inhibitor. Together, these data demonstrate, for the first time, the sphingolipid regulation of nuclear import that defines and expands the adaptive capacity of the nucleocytoplasmic transport machinery.

Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity

The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na++K+)ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca2+-dependent) and (Ca2+-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca2+-dependent protein kinase stimulated the (Na++K+)ATPase activity, while activation of the Ca2+-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na++K+)ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na++K+)ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na++K+)ATPase activity in this trypanosomatid. Modulation of the (Na++K+)ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite.

γ-Hydroxybutyrate induces cyclic AMP-responsive element-binding protein phosphorylation in mouse hippocampus: An involvement of GABAB receptors and cAMP-dependent protein kinase activation

γ-Hydroxybutyrate is a widely used recreational drug. Its abuse has been associated with cognitive impairments and development of tolerance and dependence. However, the neural mechanisms underlying these effects remain unclear. In the present study we investigated the possible cellular signaling mechanisms that might mediate γ-hydroxybutyrate’s action. Acute administration of γ-hydroxybutyrate (500 mg/kg, i.p.) was found to cause a rapid and long-lasting increase in the phosphorylation level of the cAMP-responsive element-binding protein in mouse (C57/BL6) hippocampus. Pretreatment with the specific GABAB receptor antagonist [3-[1-(R)-[(3-cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxy-propyl]amino]ethyl]-benzoic acid (20 mg/kg, i.p.) prevented the action of γ-hydroxybutyrate, confirming a GABAB receptor-mediated mechanism. In addition, acute γ-hydroxybutyrate administration induced a significant increase in cytosolic cAMP-dependent protein kinase activity in the hippocampus, and pretreatment with the cAMP-dependent protein kinase inhibitor H-89 could prevent the effect of γ-hydroxybutyrate on cAMP-responsive element-binding protein phosphorylation, indicating a direct involvement of cAMP-dependent protein kinase in γ-hydroxybutyrate-induced cAMP-responsive element-binding protein phosphorylation. On the other hand, the increased expression of phosphorylated cAMP-responsive element-binding protein was not observed in the hippocampus of mice subjected to repeated γ-hydroxybutyrate exposure, suggesting the development of a γ-hydroxybutyrate-induced desensitization of the signaling pathway leading to cAMP-responsive element-binding protein activation. Since cAMP-responsive element-binding protein activation has been implicated in a variety of neural plasticities, our findings may have revealed a new mechanism underlying γ-hydroxybutyrate-induced neuroadaptations.

C4光合成におけるホスホエノールピルビン酸カルボキシラーゼ（PEPC）制御的リン酸化：細胞質におけるPEPCキナーゼのレドックス制御

C4光合成におけるホスホエノールピルビン酸カルボキシラーゼ（PEPC）制御的リン酸化：細胞質におけるPEPCキナーゼのレドックス制御

Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in Rac2-Deficient Neutrophils.

Rac2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that Rac2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on Rac2-regulated NADPH oxidase activity. Superoxide production in rac2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in rac2−/ − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in rac2−/ − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in rac2−/ − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in rac2−/ − neutrophils, we measured protein expression and activity of cytosolic phospholipase A2 (cPLA2) and protein kinase C (PKC). The expression of cPLA2 and PMA-stimulated release of AA was similar between wild-type and rac2−/ − neutrophils, suggesting that defects in AA production by PMA-stimulated rac2−/ − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47phox and p67phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or rac2−/ − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in rac2−/ − neutrophils independent of the effects on translocation of p47phox and p67phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.

Rapid non‐genomic activation of cytosolic cyclic AMP‐dependent protein kinase activity and [Ca2+]i by 17β‐oestradiol in female rat distal colon

1. In this study, the effect of 17beta-oestradiol on adenosine 3' : 5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PKA) activity was investigated. 2. Rapid (within 15 min) activation of basal PKA activity was observed in cytosolic fractions by 17beta-oestradiol but not by 17alpha-oestradiol, progesterone or testosterone. This stimulation was abolished by the specific PKA inhibitor PKI but not by the classical oestrogen receptor antagonist tamoxifen. 3. 17beta-Oestradiol did not stimulate basal PKA activity in membrane fractions or in cytosolic fractions from male rats. 4. The increase in cytosolic PKA activity was indirect as (i) it was inhibited by the adenylyl cyclase inhibitor SQ22536, (ii) it was mimicked by forskolin and (iii) 17beta-oestradiol did not cause a stimulation of basal PKA activity in either type I or type II commercially available PKA holoenzymes. 5. Protein kinase Cdelta (PKCdelta) was directly activated by 17beta-oestradiol. The specific PKC inhibitor, bisindolylmaleimide I (GF 109203X), abolished the 6. 17beta-oestradiol-induced PKA activation. 17beta-Oestradiol stimulate an increase in free intracellular calcium ion concentration ([Ca(2+)](i)) in isolated female but not male rat colonic crypts. This was inhibited by verapamil, nifedipine and zero extracellular [Ca(2+)] but unaffected by tamoxifen. 17alpha-Oestradiol, testosterone and progesterone failed to increase [Ca(2+)](i). 7. PKC and PKA inhibitors abolished the 17beta-oestradiol-induced increase in [Ca(2+)](i). 8. These results demonstrate the existence of a novel 17beta-oestradiol-specific PKA and Ca(2+) signalling pathway, which is both sex steroid- and gender-specific, in rat distal colonic epithelium.

O-120 - Inhibitory effect of regucalcin on Ca2+ - dependent protein kinase activity in rat brain cytosol: Involvement of endogenous regucalcin

O-120 - Inhibitory effect of regucalcin on Ca2+ - dependent protein kinase activity in rat brain cytosol: Involvement of endogenous regucalcin

Inhibitory effect of regucalcin on Ca 2+-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin

The effect of regucalcin, a Ca 2+-binding protein, on Ca 2+-dependent protein kinase activity in the brain cytosol of rats with different ages (5 and 50 weeks old) was investigated. The addition of calmodulin (10 μg/ml) or dioctanoylglycerol (5 μg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl 2 (1 mM), indicating that Ca 2+ calmodulin or protein kinase C is present in the cytosol. Such an increase was completely prevented by the addition of regucalcin (10 −7 M). Moreover, regucalcin (10 −7 M) significantly inhibited cytosolic protein kinase activity without Ca 2+/calmodulin or dioctanoylglycerol addition. Meanwhile, the presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) in the enzyme reaction mixture caused a significant elevation of protein kinase activity, suggesting an inhibitory effect of endogenous regucalcin. Brain cytosolic protein kinase activity was significantly elevated by increasing age (50-week-old rats). Also, regucalcin (10 −7 M) significantly decreased protein kinase activity without Ca 2+ addition in the brain cytosol of aged rats. However, the effect of anti-regucalcin monoclonal antibody (50 ng/ml) in elevating protein kinase activity was not seen in the brain cytosol of aged rats. These results suggest that regucalcin has an inhibitory effect on Ca 2+-dependent protein kinase activity in rat brain cytosol, and that the effect of endogenous regucalcin may be weakened in the brain cytosol of aged rats.

Cholecystokinin-Evoked Ca2+ Waves in Isolated Mouse Pancreatic Acinar Cells Are Modulated by Activation of Cytosolic Phospholipase A2, Phospholipase D, and Protein Kinase C

We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca2+ signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8-induced Ca2+ signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca2+ waves elicited by stimulation of high-affinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a.CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca2+ release was the same at both CCK-8 concentrations, suggesting that the secondary Ca2+ release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca2+ waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a.CCK-R are linked to phospholipase C (PLC) and phospholipase A2 (PLA2) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA2 and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca2+ wave propagation by yet unknown mechanisms.

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O 2 ·− release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an ic 50 value of 60.7 ± 8.2 μM. A comparable effect was also demonstrated in the inhibition of O 2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O 2 ·− generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP 3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca 2+] i, but it accelerated the rate of [Ca 2+] i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8 R,9 S,11 S)-(−)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7b,11a-triazadibenzo[ a, g]cycloocta[ cde]trinden-1-one, (KT 5720), a PKA inhibitor, the inhibition of O 2 ·− generation by YT-1, as well as by prostaglandin E 1 (PGE 1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE 1. Moreover, the ability of PGE 1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.

Moderate Dietary Protein and Energy Restriction Modulate cAMP-Dependent Protein Kinase Activity in Rat Liver

Very low protein diets result in a desensitization of hepatic cAMP signaling in rats, which is characterized by a loss of cAMP-dependent protein kinase (PKA) activity and type I regulatory subunit (RI). Here we have tested whether more moderate protein restriction (Trial 1) or energy restriction (Trial 2) also modulates hepatic PKA quantity and activity. In trial 1, weanling rats were allowed free access to diets containing normal protein (15%, AL-NP), moderately restricted protein (12.5%, AL-MP) and low protein (7.5%, AL-LP); in trial 2, rats were allowed free access to diet containing 15% (AL-NP) or 0.5% protein (very low protein, AL-VLP) or were energy restricted by pair-feeding a diet isonitrogenous to AL-NP but at 65% of the energy intake (ER-IN) for 14 d. Body weights were lower (P < 0.05) by d 14 in all restricted groups compared with the AL-NP group. The quantity of cytosolic RI was lower (P < 0.05) in AL-LP and AL-VLP, but not in AL-MP or ER-IN, compared with AL-NP. In contrast, there was no effect of diet on RI in the particulate fraction. RII was not changed by moderate and low protein diets in either the cytosol or particulate fraction. However, type II regulatory subunit (RII) was greater in the cytosol of ER-IN and in the particulate fraction of AL-VLP (P < 0.05) compared with AL-NP. Specific activity of PKA was lower in the cytosol and particulate fraction (P < 0.05) in the AL-VLP and ER-IN groups compared with the AL-NP group. In contrast, specific activity of PKA was maintained in cytosol from AL-LP, but lower in the particulate fraction (P < 0.05) compared with AL-NP. In summary, protein restricted-diets lower RI subunit in the cytosol; however, only in rats fed very low protein diets is this loss of RI associated with lower cytosolic PKA activity. In contrast, energy restriction lowers PKA activity in the cytosol and particulate fractions, independent of signficant reduction in RI or RII subunits. Taken together, these data indicate that moderate protein and energy restrictions have differential effects on activity and quantity of PKA.

Investigation of the inhibitory effect of broussochalcone A on respiratory burst in neutrophils

Broussochalcone A, a prenylated chalcone isolated from Broussonetia papyrifera (L.) VENT. ( Moraceae), inhibited O 2 consumption in formylmethionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC 50 values of 70.3±4.9 and 63.9±7.1 μM, respectively. Broussochalcone A did not affect the fMLP-induced increase of cellular inositol trisphosphate (IP 3) and [Ca 2+] i. However, the enzyme activity of neutrophil cytosolic protein kinase C was effectively suppressed by broussochalcone A. Broussochalcone A had no effect on either [ 3H]phorbol 12,13-dibutyrate ([ 3H]PDB) binding to neutrophil cytosolic protein kinase C or on PMA-induced membrane translocation of protein kinase C-β in neutrophils. Broussochalcone A suppressed the enzyme activity of trypsin-treated rat brain protein kinase C in a concentration-dependent manner. In PMA-activated neutrophil particulate NADPH oxidase, broussochalcone A attenuated superoxide anion radical (O 2 ⋅−) generation with an IC 50 value of 61.8±5.4 μM. These results show that the inhibitory effect of broussochalcone A on respiratory burst in neutrophils is not mediated by the reduction of phospholipase C activity, but is mediated partly by the suppression of protein kinase C activity through interference with the catalytic region and by the attenuation of O 2 ⋅− generation from the NADPH oxidase complex.

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in rat renal cortex cytosol.

The effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl2 (0.5 mM) plus calmodulin (10 microg/ml) in the enzyme reaction mixture . This increase was completely prevented by the addition of trifluoperazine (25 microM), an antagonist of calmodulin. The cytosolic Ca2+/calmodulin-dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 microM. The cytosolic Ca2+/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca2+ (100-1000 microM). This increase was markedly blocked by the presence of regucalcin (0.1 microM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 microg/ml). These results demonstrate that regucalcin can regulate Ca2+/calmodulin-dependent protein kinase activity in renal cortex cells.

Extracellular Zinc Ions Induces Mitogen-Activated Protein Kinase Activity and Protein Tyrosine Phosphorylation in Bombesin-Sensitive Swiss 3T3 Fibroblasts

The growth factor-like effect of zincin vitroandin vivo,which has long been recognized was investigated with respect to its mechanisms of action. Addition of zinc chloride to bombesin-sensitive Swiss 3T3 mouse fibroblasts induced a fourfold stimulation in the cytosolic myelin basic protein kinase activity. The response was dose- and time-dependent, with an ED50of around 100 μMand a peak at 5 min. The kinase activity coeluted with p42 MAP kinase using chromatography on Mono-Q ion exchanger. Intracellular loading of cells with the heavy metal chelator BTC-5N did not attenuate the response to zinc. The action of zinc was not suppressed by long-term pretreatment with 4β-phorbol dibutyrate (48 h). Addition of 0.3 mMvanadate alone did not increase the kinase activity, but prolonged the action of zinc when added simultaneously. Addition of zinc (0.3 mM) or epidermal growth factor for 1 min resulted in a marked increase in tyrosine phosphorylation of proteins with apparent molecular weights of ≈100, 105–120, 215, and 240 kDa in whole cell extracts. Immunoprecipitation against the p85 subunit of phosphatidylinositol 3-kinase resulted in the appearance of two phosphotyrosine-containing proteins, 100 and 115 kDa, in extracts from cells treated with zinc or epidermal growth factor, indicating that the tyrosine phosphorylation was recognized by the corresponding SH2-domains. The present study demonstrates that extracellular zinc has the potential to partially mimic the action of growth factors on intracellular MAP kinase activation and protein tyrosine phosphorylation.

Endotoxin-induced alterations in rat colonic water and electrolyte transport

This study examines the effects of endotoxin on intestinal water and electrolyte transport in adult male rats. Endotoxin (1.55 mg/kg, intravenously) reduced in vivo colonic saline absorption 61% in 1 hour. In vitro unidirectional and net 22Na and 36Cl fluxes showed that endotoxin significantly decreased net colonic 22Na absorption compared with control colons (0.3 ± 1.7 vs. 4.8 ± 1.1 μEq/h × cm2). Although endotoxin had no significant effect on basal short circuit current (Isc) and conductance, 3H-inulin flux studies suggested an increase in colonic permeability. Isc responses to the 5′-cyclic adenosine monophosphate (cAMP)-dependent secretagogues prostaglandin E2 (1 μmol/L) and vasoactive intestinal peptide (0.1 μmol/L) were diminished by 80% and 50%, respectively. However, cytosolic cAMP-dependent protein kinase activity under basal and stimulated (6 μmol/L 8-bromo-cAMP) conditions was not altered by endotoxin treatment. The Isc responses to 10 μmol/L bethanechol, a Ca2+-dependent agonist, were not effected by endotoxin treatment. It was concluded that endotoxin significantly affects colonic transport function and may contribute to the development of diarrhea in inflammatory bowel diseases.