Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism

Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-alpha, -delta, and -epsilon were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-alpha leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-alpha activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.

Extracellular nucleotides induce migration of renal mesangial cells by upregulating sphingosine kinase-1 expression and activity

BACKGROUND AND PURPOSE: Extracellular nucleotides act as potent mitogens for renal mesangial cells (MC). In this study we determined whether extracellular nucleotides trigger additional responses in MCs and the mechanisms involved. EXPERIMENTAL APPROACH: MC migration was measured after nucleotide stimulation in an adapted Boyden-chamber. Sphingosine kinase-1 (SK-1) protein expression was detected by Western blot analysis and mRNA expression quantified by real-time PCR. SK activity was measured by an in vitro kinase assay using sphingosine as substrate. KEY RESULTS: Nucleotide stimulation caused biphasic activation of SK-1, but not SK-2. The first peak occurred after minutes of stimulation and was followed by a second delayed peak after 4-24 h of stimulation. The delayed activation of SK-1 is due to increased SK-1 mRNA steady-state levels and de novo synthesis of SK-1 protein, and depends on PKC and the classical MAPK cascade. To see whether nucleotide-stimulated cell responses require SK-1, we selectively depleted SK-1 from cells by using small-interference RNA (siRNA). MC migration is highly stimulated by ATP and UTP; this is mimicked by exogenously added S1P. Depletion of SK-1 by siRNA drastically reduced the effect of ATP and UTP on cell migration but not on cell proliferation. Furthermore, MCs isolated from SK-1-deficient mice were completely devoid of nucleotide-induced migration. CONCLUSIONS AND IMPLICATIONS: These data show that extracellular nucleotides besides being mitogenic also trigger MC migration and this cell response critically requires SK-1 activity. Thus, pharmacological intervention of SK-1 may have impacts on situations where MC migration is important such as during inflammatory kidney diseases.

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F(1)F(o)-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F(1)F(o)-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F(1)F(o)-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F(1)F(o)-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F(1) catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F(1) to the lipid monolayer and the F(o) membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F(o) by electron microscopy and AFM.

Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2

In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.

Mechanotransduction in striated muscle via focal adhesion kinase

Contractile tissues demonstrate a pronounced capacity to remodel their composition in response to mechanical challenges. Descriptive evidence suggests the upstream involvement of the phosphotransfer enzyme FAK (focal adhesion kinase) in the molecular control of load-dependent muscle plasticity. Thereby FAK evolves as a myocellular transducer of mechanical signals towards downstream transcript expression in myofibres. Recent advances in somatic gene therapy now allow the exploration of the functional involvement of this enzyme in mechanotransduction in intact muscle.

Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when > 100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-monomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO(X) exposure such as inflammation and smoking.

Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.

Gender-specific usage of intramyocellular lipids and glycogen during exercise

PURPOSE: Gender-specific differences in substrate utilization during exercise have been reported, typically such that women rely more on fat than men. This study investigated whether gender differences exist in the utilization of intramyocellular lipids (IMCL) and glycogen. METHODS: IMCL and glycogen, as well as total fat and carbohydrate (CHO) oxidation were measured in nine males and nine females before, during, and after an endurance exercise. The trained subjects exercised on a bicycle ergometer at 50% maximal workload for 3 h. IMCL and glycogen were determined in the thigh by magnetic resonance spectroscopy. Oxygen uptake (VO(2)) and carbon dioxide production were determined by open circuit spirometry to calculate total fat and CHO oxidation. Relative power output, percent of maximum heart rate, VO(2peak), and respiratory exchange ratio were the same. RESULTS: Average fat oxidation was the same, whereas CHO oxidation was significantly higher in males compared with females. The relative contribution of these fuels to total energy used were similar in males and females. Males and females depleted IMCL and glycogen significantly (P < 0.001) during the 3-h exercise. IMCL levels at rest (P < 0.05) and its depletion during exercise (P < 0.001) were significantly higher in males compared with females, whereas glycogen was stored and used in the same range by both genders. CONCLUSION: During this 3-h exercise, energy supplies from fat and CHO were similar in both genders, and males as well as females reduced their IMCL stores significantly. The larger contribution of IMCL during exercise in males compared with females could either be a result of gender-specific substrate selection, or different long-term training habit.

Cupiennin 1a, an antimicrobial peptide from the venom of the neotropical wandering spider Cupiennius salei, also inhibits the formation of nitric oxide by neuronal nitric oxide synthase

Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2) is a potent venom component of the spider Cupiennius salei. Cupiennin 1a shows multifaceted activity. In addition to known antimicrobial and cytolytic properties, cupiennin 1a inhibits the formation of nitric oxide by neuronal nitric oxide synthase at an IC50 concentration of 1.3 +/- 0.3 microM. This is the first report of neuronal nitric oxide synthase inhibition by a component of a spider venom. The mechanism by which cupiennin 1a inhibits neuronal nitric oxide synthase involves complexation with the regulatory protein calcium calmodulin. This is demonstrated by chemical shift changes that occur in the heteronuclear single quantum coherence spectrum of 15N-labelled calcium calmodulin upon addition of cupiennin 1a. The NMR data indicate strong binding within a complex of 1 : 1 stoichiometry.

Alterations in nitric oxide synthase isoforms in acute lower limb ischemia and reperfusion

Alterations in nitric oxide synthase (NOS) are implicated in ischemia and ischemia-reperfusion injury. Changes in the 3 NOS isoforms in human skeletal muscle subjected to acute ischemia and reperfusion were studied. Muscle biopsies were taken from patients undergoing total knee replacement. Distribution of the specific NOS isoforms within muscle sections was studied using immunohistochemistry. NOS mRNA levels were measured using real-time reverse transcription-polymerase chain reaction and protein levels studied using Western blotting. NOS activity was also assessed using the citrulline assay. All 3 NOS isoforms were found in muscle sections associated with muscle fibers and microvessels. In muscle subjected to acute ischemia and reperfusion, NOS I/neuronal NOS mRNA and protein were elevated during reperfusion. NOS III/endothelial NOS was also upregulated at the protein level during reperfusion. No changes in NOS II/inducible NOS expression or NOS activity occurred. In conclusion, alterations in NOS I and III (neuronal NOS and endothelial NOS) at different levels occurred after acute ischemia and reperfusion in human skeletal muscle; however, this did not result in increased NOS activity. In the development of therapeutic agents based on manipulation of the NO pathway, targeting the appropriate NOS isoenzymes may be important.

Inducible nitric oxide synthase and the effect of aminoguanidine in experimental neonatal meningitis

This study explored the role of inducible nitric oxide (NO) synthase (iNOS) in an infant rat model of group B streptococcal meningitis. Brain iNOS activity increased during meningitis (P < .001), and iNOS was detected by immunocytochemistry in the walls of meningeal vessels and cells of the cerebrospinal fluid (CSF) inflammation. Animals treated with iNOS inhibitor aminoguanidine (AG; 130 mg/kg every 8 h) had reduced NO production (P < .05), higher CSF bacterial titers (P < .05), and increased incidence of seizures (P < .01) compared with untreated infected animals. AG also increased areas of severe hypoperfusion in the cortex (31% +/- 14% in controls vs. 56% +/- 16% in AG; P < .01) and the extent of cortical neuronal injury, both when administered at the time of infection (P < .05) and in established meningitis (P < .02). Thus, NO produced by iNOS may be beneficial in this model of experimental meningitis by reducing cerebral ischemia.

IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils

The contribution of basophils in allergic disease and other Th2-type immune responses depends on their persistence at sites of inflammation, but the ligands and molecular pathways supporting basophil survival are largely unknown. The comparison of rates of apoptosis and of the expression of antiapoptotic proteins in different human granulocyte types revealed that basophils have a considerably longer spontaneous life span than neutrophils and eosinophils consistent with high levels of constitutive Bcl-2 expression. Interleukin-3 (IL-3) is the only ligand that efficiently protects basophils from apoptosis as evidenced by screening a large number of stimuli. IL-3 up-regulates the expression of the antiapoptotic proteins cIAP2, Mcl-1, and Bcl-X(L) and induces a rapid and sustained de novo expression of the serine/threonine kinase Pim1 that closely correlates with cytokine-enhanced survival. Inhibitor studies and protein transduction of primary basophils using wild-type and kinase-dead Pim1-Tat fusion-proteins demonstrate the functional importance of Pim1 induction in the IL-3-enhanced survival. Our data further indicate that the antiapoptotic Pim1-mediated pathway operates independently of PI3-kinase but involves the activation of p38 MAPK. The induction of Pim1 leading to PI3-kinase-independent survival as described here for basophils may also be a relevant antiapoptotic mechanism in other terminally differentiated leukocyte types.