Inhibition by EGTA of the formation of a biofilm by clinical strains of Staphylococcus aureus.

The effect of EGTA on the adhesion and on the formation of a biofilm by two reference and eight clinical strains of Staphylococcus aureus was studied. All the clinical strains were isolated from patients from Kinshasa. Spa typing confirmed that these clinical strains were distinct. The Biofilm Ring Test (BFRT®) showed that EGTA (100 µM-10 mM) inhibited the adhesion of the four clinical methicillin-resistant (MRSA) strains and the crystal violet staining method that it inhibited the formation of a biofilm by all the strains. Divalent cations abolished the effect of EGTA on the formation of a biofilm, specially in the clinical MRSA strains. EGTA had no effect on established biofilms. Only concentrations of EGTA higher than 10 mM were toxic to eukaryotic cells. Our results establish the effectiveness and the safety of lock solutions with EGTA to prevent the formation in vitro of biofilms by S. aureus.

Contribution of the production of quormones to some phenotypic characteristics of Pseudomonas aeruginosa clinical strains.

The contribution of quorum sensing in some phenotypic and pathogenic characteristics of Pseudomonas aeruginosa was studied. The production of acylhomoserine lactones (AHL) by planktonic cultures of eight clinical and reference strains of P. aeruginosa was evaluated using two biosensors. The adhesion of the bacteria on a surface (Biofilm Ring Test ®, BFRT), their capacity to develop a biofilm (crystal violet staining method, CVSM), their sensitivity to tobramycin and their secretion of proteases or of rhamnolipids were also measured. The production and the release of AHL widely varied among the eight strains. An analysis of the extracts by TLC showed that 3-oxo-C8-HSL, 3-oxo-C10-HSL and 3-oxo-C12-HSL were released by the five strains producing the highest amount of Cn≥6-HSL. The genes lasI and lasR involved in the synthesis and response to 3-oxo-C12-HSL were detected in the genomes of all strains. Two clinical strains had deletions in the lasR gene leading to truncation of the protein. One subpopulation of the PAO1 strain had a major deletion (98 bp) of the lasR gene. Strains with significant mutations of lasR secreted the lowest amount of AHL, probably due to deficiencies in the self-induction and amplification of the synthesis of the lactone. These strains formed a biofilm with low biomass. C4-HSL production also differed among the strains and was correlated with rhamnolipid production and biofilm formation. Whereas the production of AHL varied among P. aeruginosa strains, few correlations were observed with their phenotypic properties except with their ability to form a biofilm.

Activation of calcium-insensitive phospholipase A(2) (iPLA(2)) by P2X(7) receptors in murine peritoneal macrophages.

Free fatty acid releases are triggered by PLA2 activation and are substrates for many enzymes such as cyclooxygenases. These reactions are responsible for the production of many prostaglandins implicated in the inflammation yet many purinergic receptors have been implicated in diseases characterised by chronic inflammation. The role of P2X receptors was evaluated in LPS-primed murine peritoneal macrophages which were labelled with either [(3)H]-oleic acid or [(3)H]-arachidonic acid. Ten µmolar thapsigargin and 1mM ATP stimulated the release of both unsaturated acids. ATP had no effect at 10 µM and ivermectin had no effect on the response to ATP. The response to ATP was inhibited by magnesium and was not observed with cells from P2X(7)(-/-) mice. The response to ATP was not affected by the removal of extracellular calcium and was inhibited by arachidonyltrifluoromethyl ketone and bromoenol lactone but not by pyrrophenone. The release of the [(3)H]-fatty acids by ATP and thapsigargin was diminished by PD-98058, an inhibitor of MEK-1. It was concluded that in LPS-primed macrophages, P2X(7) receptors, not P2X(4) receptors, activated an iPLA(2) and promoted the release of unsaturated fatty acids secondary to the activation of a kinase. This response might contribute to the inflammation provoked by extracellular ATP.

Identification of peptides derived from the human antimicrobial peptide LL-37 active against biofilms formed by Pseudomonas aeruginosa using a library of truncated fragments.

Persistent Pseudomonas aeruginosa infections are a major cause of morbidity and mortality in cystic fibrosis (CF) patients and are linked to the formation of a biofilm. The development of new biofilm inhibition strategies is thus a major challenge. LL-37 is the only human antimicrobial peptide derived from cathelicidin. The effects on the P. aeruginosa PAO1 strain of synthetic truncated fragments of this peptide were compared with the effects of the original peptide. Fragments of LL-37 composed of 19 residues (LL-19, LL13-31, and LL7-25) inhibited biofilm formation. The strongest antibiofilm activity was observed with the peptides LL7-37 and LL-31, which decreased the percentage of biomass formation at a very low concentration. Some peptides were also active on the bacteria within an established biofilm. LL7-31, LL-31, and LL7-37 increased the uptake of propidium iodide (PI) by sessile bacteria. The peptide LL7-37 decreased the height of the biofilm and partly disrupted it. The peptides active within the biofilm had an infrared spectrum compatible with an α-helix. LL-37, but not the peptides LL7-31 and LL7-37, showed cellular toxicity by permeabilizing the eukaryotic plasma membrane (uptake of ethidium bromide and release of lactate dehydrogenase [LDH]). None of the tested peptides affected mitochondrial activity in eukaryotic cells. In conclusion, a 25-amino-acid peptide (LL7-31) displayed both strong antimicrobial and antibiofilm activities. The peptide was even active on cells within a preformed biofilm and had reduced toxicity toward eukaryotic cells. Our results also suggest the contribution of secondary structures (α-helix) to the activity of the peptides on biofilms.

Effect of pluronic acid F-127 on the toxicity towards eukaryotic cells of CSA-13, a cationic steroid analogue of antimicrobial peptides.

AIMS: CSA-13 is an antimicrobial cationic steroid with some toxicity against eukaryotic cells. The purpose of this work was to test whether pluronic acid F-127 could interfere with the toxicity of CSA-13 on human umbilical vein endothelial (HUVEC) without modifying its bactericidal activity against Pseudomonas aeruginosa. METHODS AND RESULTS: The addition of pluronic acid F-127 slightly decreased the number of dead cells after exposure to CSA-13. Pluronic acid F-127 blocked the permeabilizing effect of CSA-13 on the plasma membrane of HUVEC (uptake of ethidium bromide, release of lactate dehydrogenase) without modifying its toxic effect on their mitochondrial function (MTT test, uptake of tetramethyl rhodamine ethyl ester). CONCLUSION: Pluronic acid F-127 decreased the toxicity of CSA-13 against eukaryotic cells without completely protecting them from mitochondrial damage at high concentrations of the drug. SIGNIFICANCE AND IMPACT OF THE STUDY: This work establishes that studies on the toxic effects of synthetic antimicrobials on eukaryotic cells should not only focus on the permeability of the plasma membrane but also on the integrity of the mitochondria.

Distinct regulation by lipopolysaccharides of the expression of interleukin-1ß by murine macrophages and salivary glands.

The regulation of interleukin (IL)-1 expression and secretion by salivary glands and macrophages in response to lipopolysaccharides (LPS) was compared. In wild-type mice, injection of LPS significantly decreased the volume of saliva stimulated by pilocarpine and increased its protein and amylase concentration. It did not modify the salivary concentration of IL-1ß. The cytokine was expressed by submandibular acini and ducts. Macrophages also expressed IL-1ß but at lower concentration than salivary glands. The pre-incubation of macrophages with LPS increased the phosphorylation of IκB and the expression of IL-1ß. Adenosine triphosphate also promoted the secretion of the cytokine by these cells. These responses were absent in submandibular gland cells. These glands expressed CD14, TLR4 and MyD88. P2X(7)-KO mice secreted a lower volume of saliva which contained less proteins and amylase. In conclusion, IL-1ß is constitutively expressed by submandibular glands and its secretion is not regulated by a P2X(7) agonist. In these cells, LPS do not activate the nuclear factor-κB-pro-IL-1ß axis in spite of the expression of the proteins involved in their recognition.

Study of the formation of a biofilm by clinical strains of Staphylococcus aureus.

A study on biofilm formation was carried out using five methicillin-sensitive [MSSA] and five methicillin-resistant [MRSA] strains of S. aureus. In each group, there were four strains isolated from patients from Kinshasa (Democratic Republic of Congo, DRC) and one reference strain. All of the strains were hydrophobic. The adherence of the bacteria to an abiotic surface was studied with the Biofilm Ring Test (BFRT®) and the crystal violet staining method (CVSM). Both techniques showed that eight of the strains formed biofilms within 2-3 h. The extent of the biofilm formed by one strain could only be observed with the CVSM. Periodate prevented the formation of biofilms and, in separate experiments, destroyed the biofilm pre-formed by the MSSA reference, but not those pre-formed by the clinical strains. Proteinase K destroyed all pre-formed biofilms. Six of the strains were icaA+; the clinical MSSA strains were not. The results also indicated different mechanisms of biofilm development between MSSA and MRSA clinical strains. The BFRT® and the CVSM are complementary techniques to study the adhesion of bacteria and the development of biofilms.

Effect of a low concentration of a cationic steroid antibiotic (CSA-13) on the formation of a biofilm by Pseudomonas aeruginosa.

AIMS: Cationic steroids like CSA-13 have been designed by analogy with antimicrobial cationic peptides and have bactericidal properties. The purpose of this work was to evaluate the effect of a low concentration (1 mg l(-1)) of CSA-13 on the formation of a biofilm by eight strains of Pseudomonas aeruginosa (four mucoid and four nonmucoid strains) on an inert surface. METHOD AND RESULTS: The biofilm formation was measured with the Crystal Violet method. CSA-13 inhibited the formation of a biofilm by three strains. The zeta potential varied among the strains. The inhibition by the cationic steroid analogue affected the populations of bacteria with the lowest zeta potential. P. aeruginosa bound a fluorescent, more hydrophobic analogue of CSA-13 but there was no correlation between this binding and the inhibition by CSA-13 of biofilm formation. The interaction of CSA-13 with bacteria did not modify their ability to produce rhamnolipids. CONCLUSIONS: A low concentration of CSA-13 inhibits the formation of a biofilm by P. aeruginosa through electrostatic interactions and without affecting the production of rhamnolipids. SIGNIFICANCE AND IMPACT OF THE STUDY: A low, nontoxic concentration of CSA-13 might be beneficial for the prevention of biofilm formation.

Stimulation by P2X7 receptors of calcium-dependent production of reactive oxygen species (ROS) in rat submandibular glands.

BACKGROUND: Agonists of P2X7 receptors increase the production of reactive oxygen species (ROS) in immunocytes. In this work we tested this response and its effect on mitochondrial inner membrane potential (Deltapsi(m)) in exocrine glands. METHODS: The production of ROS by rat submandibular glands was investigated by measuring the oxidation of dichlorodihydrofluorescein (DCFH), a fluorescent probe. The Deltapsi(m) was estimated with tetramethylrhodamine. RESULTS: Activation of P2X7 receptors by ATP or Bz-ATP increased the production of ROS. This response was not modified by inhibitors of phospholipase A2 or of various kinases. The effect of ATP was calcium-dependent and was blocked by diphenyliodonium, an inhibitor of flavoproteins. It was not affected by rotenone, an inhibitor of the complex I of the mitochondrial electron transfer chain. Scavengers of ROS had no effect on the dissipation of Deltaψ(m) by ATP. CONCLUSIONS: We conclude that, in rat submandibular glands, P2X7 receptors stimulate in a calcium-dependent manner an oxidase generating ROS, suggesting the involvement of the dual oxidase Duox2. The production of ROS does not contribute to the depolarization of mitochondria by purinergic agonists. GENERAL SIGNIFICANCE: Purinergic receptors could be regulators of the bactericidal properties of saliva by promoting both the secretion of peroxidase from acinar cells and by activating Duox2.

Study of the initial phase of biofilm formation using a biofomic approach.

AIMS: The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test and the Crystal violet staining technique. METHODS AND RESULTS: Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test and after 6h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility. CONCLUSIONS: Our results show that the BioFilm Ring Test is a method specially suited for the study of the initial phase of the formation of a biofilm. SIGNIFICANCE AND IMPACT OF STUDY: The BioFilm Ring Test is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.

Role of sodium in mitochondrial membrane depolarization induced by P2X7 receptor activation in submandibular glands.

The effect of ATP on mitochondrial membrane depolarization in rat submandibular glands was investigated. Exposure of the cell suspension to high concentrations of ATP induced a sustained depolarization of mitochondrial membrane. This effect was blocked in the presence of magnesium and reproduced by low concentrations of 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), suggesting the implication of the P2X(7) purinergic receptor. This point was confirmed by comparison of the response to ATP by wild-type and P2X(7) knock-out (P2X(7)R(-/-)) mice. Mitochondria took up calcium after ATP stimulation but the depolarization of the mitochondrial membrane by ATP was not affected by the removal of calcium from the extracellular medium. It was nearly fully suppressed in the absence of sodium and partially blocked by the mitochondrial Na/Ca exchanger inhibitor 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Both ATP and monensin increased the uptake of extracellular sodium (as shown by the depolarization of the plasma membrane) but the sodium ionophore did not affect the mitochondrial membrane potential. It is concluded that the activation of P2X(7) receptors depolarizes the mitochondrial membrane. The uptake of extracellular sodium is necessary but not sufficient to induce this response.

Cholesterol depletion perturbs calcium handling by rat submandibular glands.

The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption.

Regulation by clozapine of calcium handling by rat submandibular acinar cells.

The effect of clozapine on the intracellular concentration of calcium ([Ca2+](i)) in rat submandibular acinar cells was tested. By itself clozapine had no effect on the mobilization of intracellular pools of calcium or on the uptake of extracellular calcium. It inhibited the increase of the [Ca2+](i) in response to carbachol (half-maximal inhibitory concentrations, IC(50)=100nM) and to norepinephrine and epinephrine (IC(50)=10nM) without affecting the response to substance P, extracellular ATP or thapsigargin. Clozapine inhibited the uptake of extracellular calcium in response to epinephrine but not to substance P, ATP or thapsigargin. It also decreased the production of inositol phosphates elicited by epinephrine but not by substance P or fluoride. It is concluded that, by itself, clozapine has no effect on the [Ca2+](i) in rat salivary acinar cells. It selectively inhibits muscarinic and adrenergic receptors in the acinar plasma membrane.

Identification of a functionally important conformation-sensitive region of the secretory Na+-K+-2Cl- cotransporter (NKCC1).

The secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a member of a small gene family of electroneutral salt transporters that play essential roles in salt and water homeostasis in many mammalian tissues. We have identified a highly conserved residue (Ala-483) in the sixth membrane-spanning segment of rat NKCC1 that when mutated to cysteine renders the transporter sensitive to inhibition by the sulfhydryl reagents 2-aminoethyl methanethiosulfonate (MTSEA) and 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The mutation of Ala-483 to cysteine (A483C) results in little or no change in the affinities of NKCC1 for substrate ions but produces a 6-fold increase in sensitivity to the inhibitor bumetanide, suggesting a specific modification of the bumetanide binding site. When residues surrounding Ala-483 were mutated to cysteine, only I484C was sensitive to inhibition by MTSEA and MTSET. Surprisingly I484C showed increased transport activity in the presence of low concentrations of mercury (1-10 microm), whereas A483C showed inhibition. The inhibition of A483C by MTSEA was unaffected by the presence or absence of sodium and potassium but required the presence of extracellular chloride. Taken together, our results indicate that Ala-483 lies at or near an important functional site of NKCC1 and that the exposure of this site to the extracellular medium is dependent on the conformation of the transporter. Specifically, our results indicate that the cysteine introduced at residue 483 is only available for interaction with MTSEA when chloride is bound to NKCC1 at the extracellular surface.

Multiple effects of trichloroethanol on calcium handling in rat submandibular acinar cells.

The effect of trichloroethanol (TCEt), the active metabolite of chloral hydrate, on the intracellular concentration of calcium ([Ca(2+)](i)) was investigated in rat submandibular glands (RSMG) acini loaded with fura-2. TCEt (1 - 10 mM) increased the [Ca(2+)](i) independently of the presence of calcium in the extracellular medium. Dichloroethanol (DCEt) and monochloroethanol (MCEt) reproduced the stimulatory effect of TCEt but at much higher concentrations (about 6 fold higher for DCEt and 20 fold higher for MCEt). TCEt mobilized an intracellular pool of calcium, which was depleted by a pretreatment with thapsigargin, an inhibitor of the sarcoplasmic and endoplasmic reticulum calcium-dependent ATPases, but not with FCCP, an uncoupler of mitochondria. TCEt 10 mM inhibited by 50% the thapsigargin-sensitive microsomal Ca(2+)-ATPase. DCEt 10 mM and MCEt 10 mM inhibited the ATPase by 20 and 10%, respectively. TCEt inhibited the increase of the [Ca(2+)](i) and the production of inositol phosphates in response to carbachol, epinephrine and substance P. TCEt inhibited the uptake of calcium mediated by the store-operated calcium channel (SOCC). ATP and Bz-ATP increased the [Ca(2+)](i) in RSMG acini and this effect was blocked by extracellular magnesium, by Coomassie blue and by oxydized ATP (oATP). TCEt potentiated the increase of the [Ca(2+)](i) and of the uptake of extracellular calcium in response to ATP and Bz-ATP. TCEt had no effect on the uptake of barium and of ethidium bromide in response to purinergic agonists. These results suggest that TCEt, at sedative concentrations, exerts various effects on the calcium regulation: (1) it mobilizes a thapsigargin-sensitive intracellular pool of calcium in RSMG acini; (2) it inhibits the uptake of calcium via the SOCC; (3) it inhibits the activation by G protein-coupled receptors of a polyphosphoinositide-specific phospholipase C. It does not interfere with the activation of the ionotropic P2X receptors. The use of chloral hydrate should be avoided in studies exploring the in vivo responses to sialagogues.

Modulation by propranolol of the uptake of ethidium bromide by rat submandibular acinar cells exposed to a P2X(7) agonist or to maitotoxin.

We have compared the formation of pores in rat submandibular acinar cells in response to 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and maitotoxin. Bz-ATP (100 microM) permeabilized the cells to ethidium bromide. The uptake of ethidium increased to 29+/-1% of maximal uptake in 10 min. DL-Propranolol (300 microM) inhibited the Bz-ATP-induced uptake of ethidium bromide by 40% without affecting the P2X(7)-gated cation channel. The inhibitory effect of DL-propranolol on the formation of pores by Bz-ATP was reproduced by D-propranolol, an optical isomer with very poor beta-blocking activity. Tenidap, an antiinflammatory drug, enhanced the permeabilization in response to Bz-ATP. Propanolol inhibited the response to tenidap plus Bz-ATP. The effect of propranolol was reproduced by labetolol, a beta-adrenergic antagonist with membrane-stabilizing properties, but not by atenolol, which blocks beta-adrenergic receptors but has no effect on the stability of the membrane. In the presence of extracellular calcium, maitotoxin also increased the uptake of ethidium bromide. Tenidap had no effect on this response, which was delayed by propranolol. In conclusion, we have shown that propranolol, in a range of 10-300 microM, inhibits the pore-forming activity of the P2X(7) receptor without affecting the opening of the cation channel coupled to this receptor. This inhibition is not related to its beta-adrenergic blocking activity but rather to its membrane-stabilizing properties. Propranolol also delays the uptake of ethidium bromide in response to maitotoxin. This is in agreement with the current view that P2X(7) agonists and maitotoxin share a common pore.

Potentiation by propofol of the response of rat submandibular acinar cells to purinergic agonists.

The effect of propofol (2,6-diisopropylphenol) on the intracellular concentration of calcium ([Ca(2+)](i)) and on the response of rat submandibular acini to purinergic agonists was studied. By itself, propofol (60 to 200 microM) slowly increased the [Ca(2+)](i) without affecting the production of inositol phosphates. The increase of the [Ca(2+)](i) involved for about 50% the mobilization of thapsigargin-sensitive intracellular calcium pools. The rest of the calcium originated from a pool distinct from mitochondria. Propofol also increased the uptake of extracellular calcium but not manganese by a mechanism inhibited by nickel. The variation of the [Ca(2+)](i) by propofol provoked a decrease of cell volume measured by light scattering. Propofol increased the effect of a maximal concentration of extracellular ATP on the [Ca(2+)](i). This interaction could be observed when propofol and ATP were added simultaneously to the medium but not when propofol had been removed from the medium before adding ATP. Among ATP analogs, propofol only increased the response to benzoyl-ATP (Bz-ATP). The blockade of P2X(7) receptors with oxidized ATP or Coomassie blue did not prevent the interaction between propofol and ATP. The effect of propofol could also be observed even when the concentration of ATP(4-) was decreased by extracellular magnesium to such a level that only P2X(4) receptors could possibly be activated by the nucleotide. Propofol had no effect on the uptake of manganese, the formation of pores and the activation of phospholipase D in response to a P2X(7) agonist. These results exclude an interaction with this receptor. It is concluded that, in rat submandibular acini, propofol can increase the [Ca(2+)](i) and decrease the cell volume. Propofol can also modulate the activation of P2X(4) receptors by extracellular nucleotides. These effects are observed at concentrations of propofol reached during the induction of anesthesia and might explain why hypersalivation has been reported as one of the side-effects of propofol.

Bromoenol lactone enhances the permeabilization of rat submandibular acinar cells by P2X7 agonists.

The permeabilizing effect of P2X(7) agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. The uptake of ethidium bromide by acini incubated at 37 degrees C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin. The response to ATP was dose-dependent (half-maximal concentration around 40 microM) and it was decreased when the temperature was lowered to 25 degrees C. Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 microM). UTP or 2-methylthioATP had no effect. The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. ATP increased the activity of a calcium-insensitive phospholipase A(2) (iPLA(2)). Bromoenol lactone (BEL) inhibited the iPLA(2) stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. From these results it is concluded that the activation of P2X(7) receptors permeabilizes rat submandibular acinar cells. The pore-forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA(2) by the receptor.

Regulation of the Na+-K+(NH4+)-2Cl- cotransporter of rat submandibular glands.

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH4Cl, and the variations of the intracellular concentration of calcium ([Ca2+]i) and the intracellular pH (pHi) were measured using fura-2 and 2',7'-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein. More than 5 mmol/l NH4Cl significantly increased the [Ca2+]i without affecting the response to 100 micromol/l carbachol. When exposed to 1 and 5 mmol/l NH4Cl, the cells acidified immediately. At 30 mmol/l, NH4Cl first alkalinized the cells and the pHi subsequently dropped. This drop reflects the uptake of NH4+ ions that dissociate to NH3 and H+ in the cytosol. These protons are exchanged for extracellular sodium by the Na+/H+ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH4Cl. Ouabain partly blocked the uptake of NH4+. In the combined presence of ouabain and bumetanide (an inhibitor of the Na+-K+-2Cl- cotransporter), 1 mmol/l NH4Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na+-K+-2Cl- cotransporter in the uptake of NH4+ was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH4+ by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH4+ by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl-ATP but not by CTP, GTP, UTP, ADP, or ADPbetaS. ATP only slightly inhibited the increase of cyclic AMP (-22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the beta-adrenergic agonist. ATP increased the release of 3H-arachidonic acid from prelabeled cells but SK&F 96365, an imidazole-based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP-stimulated activity of the Na+-K+-2Cl- cotransporter.

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.