Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion

Reduced gastrointestinal HCO3- secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical HCO3- transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/HCO3- exchangers and anion conductances in basal and cAMP-stimulated duodenal HCO3- secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, HCO3- secretion rates, and 36Cl-, 22Na+, and 3H+ mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl- from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal HCO3- secretion but did not influence the HCO3- secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional 22Na+ and 36Cl- flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated HCO3- secretion. The ratio of Cl- to HCO3- in the anion secretory response was compatible with both Cl- and HCO3- being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/HCO3- exchanger mediates a significant part of basal HCO3- secretion but is not involved in the HCO3- secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl- over HCO3- in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal HCO3- secretion is directly mediated by CFTR.     

Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum.

The objective of this study was to determine whether a constitutive isoform of nitric oxide (NO) synthase is present in rabbit corpus cavernosum that could account for the involvement of the L-arginine-NO pathway in neurogenically-elicited relaxation of the corpus cavernosum and, therefore, penile erection. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO(x) (NO+nitrite [NO2-]+nitrate [NO3-]) by chemiluminescence after reduction of NO(x) to NO by acidic vanadium (III). Equimolar quantities of NO plus citrulline were generated from L-arginine and the formation of both products was time-dependent at 37 degrees C. NO synthase activity was distributed almost entirely to the cytosolic fraction. Enzymatic activity was completely dependent on NADPH, calmodulin, and calcium. Addition of tetrahydrobiopterin increased NO synthase activity by about 30 percent. The NO synthase inhibitor NG-nitro-L-arginine, abolished enzymatic activity. The Km for L-arginine was 17 microM and the Vmax of the reaction was 18 pmol/min/mg protein. These observations indicate that a cytosolic, constitutive isoform of NO synthase, like that found in brain neuronal tissue, is present in rabbit corpus cavernosum.     

Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells

Na-nutrient cotransport processes are not only important for the assimilation of essential nutrients but also for the absorption of Na in the mammalian small intestine. The effect of constitutive nitric oxide (cNO) on Na-glucose (SGLT-1) and Na-amino acid cotransport (NAcT) in the mammalian small intestine is unknown. Inhibition of cNO synthase with N(G)-nitro-l-arginine methyl ester (L-NAME) resulted in the inhibition of Na-stimulated (3)H-O-methyl-D-glucose uptake in villus cells. However, Na-stimulated alanine uptake was not affected in these cells. The L-NAME-induced reduction in SGLT-1 in villus cells was not secondary to an alteration in basolateral membrane Na-K-ATPase activity, which provides the favorable Na gradient for this cotransport process. In fact, SGLT-1 was inhibited in villus cell brush-border membrane (BBM) vesicles prepared from animals treated with L-NAME. Kinetic studies demonstrated that the mechanism of inhibition of SGLT-1 was secondary to a decrease in the affinity for glucose without a change in the maximal rate of uptake of glucose. Northern blot studies demonstrated no change in the mRNA levels of SGLT-1. Western blot studies demonstrated no significant change in the immunoreactive protein levels of SGLT-1 in ileal villus cell BBM from L-NAME-treated rabbits. These studies indicate that inhibition of cNO production inhibits SGLT-1 but not NAcT in the rabbit small intestine. Therefore, whereas cNO promotes Na-glucose cotransport, it does not affect NAcT in the mammalian small intestine.     

Effect of basolateral CO2/HCO3- on intracellular pH regulation in the rabbit S3 proximal tubule

We used the absorbance spectrum of the pH-sensitive dye dimethylcarboxyfluorescein to monitor intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule, and examined the effect on pHi of switching from a HEPES to a CO2/HCO3- buffer in the lumen and/or the bath (i.e., basolateral solution). Solutions were titrated to pH 7.40 at 37 degrees C. With 10 mM acetate present bilaterally (lumen and bath), this causing steady-state pHi to be rather high (approximately 7.45), bilaterally switching the buffer from 32 mM HEPES to 5% CO2/25 mM HCO3- caused a sustained fall in pHi of approximately 0.26. However, with acetate absent bilaterally, this causing steady-state pHi to be substantially lower (approximately 6.9), bilaterally switching to CO2/HCO3- caused a transient pHi fall (due to the influx of CO2), followed by a sustained rise to a level approximately 0.18 higher than the initial one. The remainder of the experiments was devoted to examining this alkalinization in the absence of acetate. Switching to CO2/HCO3- only in the lumen caused a sustained pHi fall of approximately 0.15, whereas switching to CO2/HCO3- only in the bath caused a transient fall followed by a sustained pHi increase to approximately 0.26 above the initial value. This basolateral CO2/HCO3(-)-induced alkalinization was not inhibited by 50 microM DIDS applied shortly after CO2/HCO3- washout, but was slowed approximately 73% by DIDS applied more than 30 min after CO2/HCO3- washout. The rate was unaffected by 100 microM bilateral acetazolamide, although this drug greatly reduced CO2-induced pHi transients. The alkalinization was not blocked by bilateral removal of Na+ per se, but was abolished at pHi values below approximately 6.5. The alkalinization was also unaffected by short-term bilateral removal of Cl- or SO4=. Basolateral CO2/HCO3- elicited the usual pHi increase even when all solutes were replaced, short or long-term (> 45 min), by N-methyl-D- glucammonium/glucuronate (NMDG+/Glr-). Luminal CO2/HCO3- did not elicit a pHi increase in NMDG+/Glr-. Although the sustained pHi increase elicited by basolateral CO2/HCO3- could be due to a basolateral HCO3- uptake mechanism, net reabsorption of HCO3- by the S3 segment, as well as our ACZ data, suggest instead that basolateral CO2/HCO3- elicits the sustained pHi increase either by inhibiting an acid-loading process or stimulating acid extrusion across the luminal membrane (e.g., via an H+ pump).     

beta -Adrenergic regulation of constitutive nitric oxide synthase in cardiac myocytes

Nitric oxide (NO) has been implicated in endogenous control ofmyocardial contractility. However, NO release has not yet been demonstrated in cardiac myocytes. Accordingly, endogenous NO production was measured with a porphyrinic microsensor positioned on the surfaceof individual neonatal or adult rat ventricular myocytes (n > 6 neonatal and adult cells perexperiment). In beating neonatal myocytes, there was no detectablespontaneous NO release with each contraction. However, norepinephrine(NE; 0.25-1 µM) elicited transient NO release from beatingneonatal (149 ± 11 to 767 ± 83 nM NO) and noncontracting adult(157 ± 13 to 791 ± 89 nM NO) cells. NO was released byadrenergic agonists with the following rank order of potency:isoproterenol(₁₂) > NE (/₁) > dobutamine (₁)  epinephrine(/₁₂) > tertbutylene (₂); NO wasnot released by phenylephrine (). NE-evoked NO release wasreversibly blocked byN^G-monomethyl-L-arginine,trifluoperazine, guanosine5'-O-(2-thiodiphosphate), andnifedipine but was enhanced by 3-isobutyl-1-methylxanthine (0.5 mM = 14.5 ± 1.6%) and BAY K 8644 (10 µM = 11.9 ± 1%). NO wasalso released by A-23187 (10 µM = 884 ± 88 nM NO), guanosine 5'-O-(3-thiotriphosphate) (1 µM = 334 ± 56 nMNO), and dibutyryl adenosine 3',5'-cyclic monophosphate(10-100 µM = 35 ± 9 to 284 ± 49 nM NO) but not by ATP,bradykinin, carbachol, 8-bromoguanosine 3',5'-cyclicmonophosphate, or shear stress. This first functional demonstration ofa constitutive NO synthase in cardiac myocytes suggests its regulationby a -adrenergic signaling pathway and may provide a novel mechanismfor the coronary artery vasodilatation and enhanced diastolicrelaxation observed with adrenergic stimulation.

     

Identification of an apical Cl(-)/HCO3(-) exchanger in the small intestine

HCO3(-) secretion is the most important defense mechanism against acid injury in the duodenum. However, the identity of the transporter(s) mediating apical HCO3(-) secretion in the duodenum remains unknown. A family of anion exchangers, which include downregulated in adenoma (DRA or SLC26A3), pendrin (PDS or SLC26A4), and the putative anion transporter (PAT1 or SLC26A6) has recently been identified. DRA and pendrin mediate Cl(-)/base exchange; however, the functional identity and distribution of PAT1 (SLC26A6) is not known. In these studies, we investigated the functional identity, tissue distribution, and membrane localization of PAT1. Expression studies in Xenopus oocytes demonstrated that PAT1 functions in Cl(-)/HCO3(-) exchange mode. Tissue distribution studies indicated that the expression of PAT1 is highly abundant in the small intestine but is low in the colon, a pattern opposite that of DRA. PAT1 was also abundantly detected in stomach and heart. Immunoblot analysis studies identified PAT1 as a approximately 90 kDa protein in the duodenum. Immunohistochemical studies localized PAT1 to the brush border membranes of the villus cells of the duodenum. We propose that PAT1 is an apical Cl(-)/HCO3(-) exchanger in the small intestine.     

Co-regulation of constitutive nitric oxide synthases and NADPH oxidase by the small GTPase Rac

Nitric oxide (NO), generated by NO synthases (NOSs), has multifarious roles in signal transduction. Reactive oxygen species (ROS), generated by ubiquitous NADPH oxidases (NOXs), also participate in cellular signaling. However, the coordination of signals conveyed by NO and ROS is poorly understood. We show that the small GTPase Rac, a component of some NOXs, also interacts with and regulates the constitutively-expressed NOSs. Cellular NO and O(2)(-) production increase or decrease together following activation or inhibition of Rac, and Rac inhibition reveals transduction mechanisms that depend upon NO (vasodilation), ROS (actin polymerization) or both (cytoskeletal organization). Thus, signaling by NO and ROS may be coordinated through a common control element.     

Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.     

Mechanism of regulation of rabbit intestinal villus cell brush border membrane Na/H exchange by nitric oxide

In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H and Cl/HCO(3) exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCl absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with l-N(G)-nitroarginine methylester (l-NAME) stimulated Na/H exchange whereas Cl/HCO(3) exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with l-NAME, Na/H exchange was also stimulated. d-NAME, an inactive analog of l-NAME, and N(6)-(1-imonoethyl)-l-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCl absorption by specifically inhibiting BBM Na/H expression.     

The CFTR associated protein CAP70 interacts with the apical Cl-/HCO3- exchanger DRA in rabbit small intestinal mucosa

DRA (down regulated in adenoma) is an intestinal anion exchanger, acting in parallel with NHE3 to facilitate ileal and colonic NaCl absorption. Furthermore it is involved in small intestinal bicarbonate secretion. Because DRA has a PDZ interaction motif, which may influence its properties, we searched for DRA-interacting PDZ adapter proteins in the small intestine. Using an overlay assay with the recombinant DRA C-terminus as a ligand, a 70 kDa protein was labeled, which was restricted to the brush border membrane in rabbit duodenal and ileal mucosa and was not detected in the colon. Destruction of the C-terminal PDZ interaction motif abolished this band, suggesting a specific protein-protein interaction. The 70 kDa protein was identified as CAP70 (CFTR associated protein of 70 kDa) by an anti-CAP70 antibody and by two in vitro binding assays after cloning CAP70 from rabbit duodenum and ileum. The interaction was recapitulated in HEK cells transfected with DRA and PDZK1, the human orthologue of CAP70. Corresponding to the overlay assay, no CAP70 mRNA or protein was detected in the colon. In vitro protein-protein interaction studies revealed specific binding of DRA to the 2nd and 3rd PDZ domain, while CFTR is known to interact with PDZ1, PDZ3, and PDZ4. The composition of macromolecular complexes assembled by CAP70 in the distal small bowel is unknown. Its restricted expression shows that it cannot be involved in NaCl absorption in the proximal colon. We suggest that CAP70 mediates regulatory functions specific to the small intestine.     

Intracellular pH regulation by HCO3-/Cl- exchange is activated during early mouse zygote development

We report here that at least one major pHi-regulatory mechanism, the HCO3-/Cl- exchanger, is quiescent in unfertilized mouse eggs but becomes fully activated during early development following fertilization. Zygotes (8-12 h postfertilization) exhibited a marked intracellular alkalinization upon external Cl- removal, which is indicative of active HCO3-/Cl- exchangers, in contrast to the very small response observed in eggs. In addition, efflux of Cl- from eggs upon external Cl- removal was much slower than that from zygotes, indicating additional pathways for Cl- to cross the plasma membrane in zygotes. Furthermore, while zygotes quickly recovered from an induced alkalosis, eggs exhibited only a slow, incomplete recovery. Following in vitro fertilization (IVF), increased HCO3-/Cl- exchanger activity was first detectable about 4 h postfertilization and reached the maximal level after about 8 h. The upregulation of HCO3-/Cl- exchanger activity after fertilization appeared to occur by activation of existing, inactive exchangers rather than by synthesis or transport of new exchangers, as the increase in activity following IVF was unaffected by inhibition of protein synthesis or by disruption of the Golgi apparatus or the cytoskeleton. This activation may depend on the Ca2+ transients which follow fertilization, as suppression of these transients, using the Ca2+ chelator BAPTA, reduced subsequent upregulation of HCO3-/Cl- exchanger activity by about 50%. Activation of pHi-regulatory systems may be a widespread feature of the earliest period of embryonic development, not restricted to species such as marine invertebrates as previously believed.     

Activation of constitutive nitric oxide synthases by oxidized calmodulin mutants

Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite.     

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl-/HCO3- exchanger, AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO2 to HCO3- + H+. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl-/HCO3- exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl-/NO3- exchange assays, which were independent of CA activity, and in Cl-/HCO3- exchange assays. Transport was measured by following changes of intracellular [Cl-] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2',7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl-/NO3- exchange activity with EC50 values in the range 0.22-2.8 microM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 microM of each compound was only 22-53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl-/HCO3- exchange assays, which depend on functional CA to produce transport substrate, 40 microM celecoxib inhibited AE1 by 62+/-4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.     

Prostaglandin synthesis by enterocyte microsomes of rabbit small intestine

We studied the prostaglandin (PG) synthetic capacity of microsomes of a relatively pure population of rabbit enterocytes and determined ideal conditions for product synthesis. The epithelial cells were freed from the basement membrane by a combination of calcium chelation and mechanical vibration, and 100,000 X g microsomes were prepared. These microsomes were found to synthesize PG from exogenously added arachidonic acid. The ideal conditions for the reaction were a microsomal protein concentration of 1.0 mg/ml, an arachidonic acid concentration of 33 uM, a reaction mixture pH of 8.0-9.5 and with epinephrine 1.5 mM added as a cofactor. The product yields increased linearly with time up to 30 min. of incubation and were inhibited by 100 uM indomethacin. Under the above ideal conditions enterocyte microsomes yielded the following products expressed as pmole/mg protein/20 min. incubation: PGF2 alpha 98 +/- 7, PGE2 48 +/- 9, PGD2 28 +/- 7, TxB2 40 +/- 5, 6 Keto PGF1 alpha 15 +/- 6.     

Prostaglandin synthesis by enterocyte microsomes of rabbit small intestine

We studied the prostaglandin (PG) synthetic capacity of microsomes of a relatively pure population of rabbit enterocytes and determined ideal conditions for product synthesis. The epithelial cells were freed from the basement membrane by a combination of calcium chelation and mechanical vibration, and 100,000 x g microsomes were prepared. These microsomes were found to synthesize PG from exogenously added arachidonic acid. The ideal conditions for the reaction were a microsomal protein concentration of 1.0 mg/ml, an arachidonic acid concentration of 33 μM, a reaction mixture pH of 8.0 − 9.5 and with epinephrine 1.5 mM added as a cofactor. The product yields increased linearly with time up to 30 min, of incubation and were inhibited by 100 μM indomethacin. Under the above ideal conditions enterocyte microsomes yielded the following products expressed as pmole/mg protein/20 min, incubation: PGF_2α 98±7, PGE₂ 48±9, PGD₂ 28±7, TxB₂ 40±5, 6 Keto PGF_1α 15 ± 6.     

The substrate anion selectivity filter in the human erythrocyte Cl-/HCO3- exchange protein, AE1

AE1 facilitates Cl-/HCO3- exchange across the erythrocyte membrane. To identify residues involved in substrate selection and translocation, we prepared an array of single cysteine mutants in an otherwise cysteineless background. These mutants spanning the C-terminal portion of the AE1 membrane domain from Phe806-Cys885 were characterized for functional activity when expressed in human embryonic kidney 293 cells by measurement of changes of intracellular pH associated with bicarbonate transport. To identify residues involved in substrate translocation, transport activity was assessed for each mutant before and after treatment with the following sulfhydryl reagents: anionic para-chloromercuibenzenesulfonate; permeant (2-aminoethyl)methanethiosulfonate; and cationic [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Among the 80 mutants, only certain key residues in the Val849-Leu863 region were inhibited by the sulfhydryl reagent, consistent with direct involvement of these sites in anion transport. In the last two transmembrane segments, only mutants in the extracellular portion of the transmembrane segments could be inhibited by sulfhydryl reagent, suggesting that the outer portions line the translocation channel and the inner portions have some other role. Sensitivity to cationic MTSET and effects of Cl- identified the substrate charge filter as Ser852-Leu857.     

Negative regulation by phosphatidylinositol 3-kinase of inducible nitric oxide synthase expression in macrophages

Triggering of the macrophage cell line RAW 264.7 with LPS promotes a transient activation of phosphatidylinositol 3-kinase (PI3-kinase). Incubation of activated macrophages with wortmannin and LY294002, two inhibitors of PI3-kinase, increased the amount of inducible nitric oxide synthase (iNOS) and the synthesis of nitric oxide. Treatment with wortmannin promoted a prolonged activation of NF-kappaB in LPS-treated cells as well as an increase in the promoter activity of the iNOS gene as deduced from transfection experiments using a 1.7-kb fragment of the 5' flanking region of the iNOS gene. Cotransfection of cells with a catalytically active p110 subunit of PI3-kinase impaired the responsiveness of the iNOS promoter to LPS stimulation, whereas transfection with a kinase-deficient mutant of p110 maintained the up-regulation in response to wortmannin. These results indicate that PI3-kinase plays a negative role in the process of macrophage activation and suggest that this enzyme might participate in the mechanism of action of antiinflammatory cytokines.