Jean-Paul Dehaye

Activation by P2X7 agonists of two phospholipases A2 (PLA2) in ductal cells of rat submandibular gland. Coupling of the calcium-independent PLA2 with kallikrein secretion.

Isolated ductal cells of rat submandibular gland phospholipid pools were labeled with [3H]arachidonic acid (AA). The tracer was incorporated preferentially to phosphatidylcholine (46% of the lipidic fraction). Extracellular ATP induced the release of [3H]AA to the extracellular medium in a time- and dose-dependent manner (EC50 = 220 μm). Among other agents tested, only 2′,3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (Bz-ATP) was able to mimic the effect of ATP (EC50 = 15 μm), without activation of phospholipase C. The purinergic antagonists oxidized ATP, suramin, and Coomassie Blue partly inhibited the response to 1 mm ATP and 100 μmBz-ATP; the response was also blocked by the addition of Mg2+ or Ni2+. Expression of P2X7receptor mRNA in these cells was confirmed by reverse transcription-polymerase chain reaction. In the presence of extracellular calcium, the phospholipase A2 inhibitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (a nonspecific inhibitor), arachidonyl trifluoromethylketone (AACOCF3, an inhibitor of the calcium-dependent cytosolic PLA2(cPLA2)), and bromoenol lactone (an inhibitor of the calcium-independent PLA2 (iPLA2)) inhibited the release of [3H]AA induced by ATP and Bz-ATP. In the absence of extracellular calcium, the release of [3H]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF3 and completely blocked by bromoenol lactone. ATP and Bz-ATP stimulated a calcium-independent secretion of kallikrein, which could be blocked by BEL but which was enhanced by AACOCF3. It is concluded that the P2X7 receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA2 and a calcium-independent iPLA2.

Identification of Peptides Derived from the Human Antimicrobial Peptide LL-37 Active against Biofilms Formed by Pseudomonas aeruginosa Using a Library of Truncated Fragments

ABSTRACT 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.

Efficacy of the Combination of Tobramycin and a Macrolide in an In Vitro Pseudomonas aeruginosa Mature Biofilm Model

ABSTRACT Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In particular, patients suffer from chronic infection due to biofilm formation by opportunistic Pseudomonas aeruginosa (32). Therefore, there is an urgent need to develop alternative ways to treat biofilm-associated clinical infections. The aim of this study was to compare the antimicrobial effects in vitro of the combinations tobramycin-clarithromycin and tobramycin-azithromycin against five P. aeruginosa biofilms and to establish the most effective combination. We performed a kinetic study over a period of 28 days of a twice-daily coadministration of the combinations tobramycin-clarithromycin and tobramycin-azithromycin on 12-day-old, mature P. aeruginosa biofilms formed on microplate pegs for 4 clinical isolates and one laboratory strain (PAO1) to simulate the treatment of CF patients with tobramycin inhalation solution (TOBI) through aerosolization. A synergy between tobramycin and clarithromycin was recorded for 3/5 biofilms, with a bacterial decrease of more than 5 log. Conversely, we found an antagonistic activity when 4 μg/ml tobramycin was administered with azithromycin at 2 μg/ml for P. aeruginosa PAO1 and with azithromycin at 2, 20, 50, 100, and 200 μg/ml for P. aeruginosa PYO1. Treatment with tobramycin at 4 μg/ml combined with clarithromycin at 200 μg/ml eradicated all five biofilms, while tobramycin-azithromycin at the same concentrations eradicated only three biofilms. Results of this study suggest that local administration of tobramycin and clarithromycin into the respiratory tract represents a better strategy than the combination tobramycin-azithromycin for the treatment of P. aeruginosa-associated pulmonary infections.

Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland.

The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X7 receptors were detected in these fractions. The location of the P2X7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-β-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with α-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca2+]i) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A2 (PLA2). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. We conclude that P2X7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA2 in response to P2X7 receptor occupancy.

Modulation by LL-37 of the responses of salivary glands to purinergic agonists.

The interaction of mice submandibular gland cells with LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a cationic peptide with immunomodulatory properties, was investigated. LL-37 at a concentration that did not affect the integrity of the cells increased the uptake of calcium and activated a calcium-insensitive phospholipase A2 (PLA2). The small release of ATP induced by LL-37 could not account for this stimulation because apyrase did not significantly block the response to LL-37. The divalent cation magnesium inhibited the response to LL-37, but this inhibition was probably nonspecific because it also inhibited the in vitro bacteriostatic effect of the peptide. The increase of calcium uptake by LL-37 was not affected by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62), a rather specific inhibitor of P2X7 receptors in mice. LL-37 also increased [Ca2+]i in cells from mice invalidated for these receptors. LL-37 had no effect on the response to carbachol. It inhibited the increase of [Ca2+]i and the activation of phospholipase D by ATP. It potentiated the activation of the PLA2 by the nucleotide. Finally, LL-37 increased the fluidity of the plasma membrane of submandibular gland cells. In conclusion, our results suggest that LL-37 is an autocrine regulator of submandibular gland cells. It does not stimulate mouse P2X7 receptors but modulates their responses.

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

The permeabilizing effect of P2X7 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°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 μM) and it was decreased when the temperature was lowered to 25°C. Benzoyl‐ATP reproduced the response to ATP (half‐maximal concentration around 10 μM). 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 A2 (iPLA2). Bromoenol lactone (BEL) inhibited the iPLA2 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 P2X7 receptors permeabilizes rat submandibular acinar cells. The pore‐forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA2 by the receptor.

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P.

The effect of extracellular ATP on the intracellular calcium concentration ([Ca2+]i) in rat submandibular glands was tested. The dose‐response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl‐ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high‐affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca2+]i in response to 100 μM carbachol. By itself, substance P (100 pM‐1 μM) increased the [Ca2+]i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM‐1 μM) increased the release of inositol trisphosphate (IP3) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI‐specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high‐affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P2Z type coupled to a non‐selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca2+]i in response to a muscarinic agonist and the activation of polyPI‐specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca2+]i.

Study of the effect of antimicrobial peptide mimic, CSA-13, on an established biofilm formed by Pseudomonas aeruginosa

The formation of a Pseudomonas aeruginosa biofilm, a complex structure enclosing bacterial cells in an extracellular polymeric matrix, is responsible for persistent infections in cystic fibrosis patients leading to a high rate of morbidity and mortality. The protective environment created by the tridimensional structure reduces the susceptibility of the bacteria to conventional antibiotherapy. Cationic steroid antibiotics (CSA)‐13, a nonpeptide mimic of antimicrobial peptides with antibacterial activity on planktonic cultures, was evaluated for its ability to interact with sessile cells. Using confocal laser scanning microscopy, we demonstrated that the drug damaged bacteria within an established biofilm showing that penetration did not limit the activity of this antimicrobial agent against a biofilm. When biofilms were grown during exposure to shear forces and to a continuous medium flow allowing the development of robust structures with a complex architecture, CSA‐13 reached the bacteria entrapped in the biofilm within 30 min. The permeabilizing effect of CSA‐13 could be associated with the death of the bacteria. In static conditions, the compound did not perturb the architecture of the biofilm. This study confirms the potential of CSA‐13 as a new strategy to combat persistent infections involving biofilms formed by P. aeruginosa.

Functional characterization of muscarinic receptors in rat parotid acini

The muscarinic agonist, carbamylcholine, stimulated amylase secretion in rat parotid acini 6-fold, the 86Rb efflux 5-fold, the 45Ca efflux 5-fold and the accumulation of inositol monophosphate, bisphosphate, trisphosphate and tetrakisphosphate 4-, 4-, 3- and 3-fold, respectively. The EC50 of carbamylcholine on these parameters were 0.4, 0.5, 1.3, 12, 12, 6 and 9 microM, suggesting spareness between phospholipase C activation and amylase secretion. These muscarinic responses were inhibited by four muscarinic antagonists with an order of potency on all parameters and on receptor occupancy (using N-[methyl-3H]scopolamine as a tracer): atropine greater than hexahydrosiladifenidol greater than pirenzepine greater than AF-DX 116. The pA2 of these antagonists on carbamylcholine-stimulated amylase secretion were 9.72 for atropine, 8.14 for hexahydrosiladifenidol, 7.16 for pirenzepine and 6.22 for AF-DX 116, indicating that the parotid muscarinic receptors were of an M2 subtype 83-fold more sensitive to hexahydrosiladifenidol than to AF-DX 116.

Binding in vitro of vasoactive intestinal peptide on isolated acini of rat parotid glands

Binding of 125I-labelled vasoactive intestinal peptide (VIP) to rat parotid acini was saturable, temperature-dependent and reversible, and reflected interaction with a single class of binding sites. Parotid glands possessed approx. 400 fmol binding sites per mg protein and binding of the tracer to these sites could be inhibited by VIP [concentration for half-maximal effect (KD), 24 nM], by the peptide histidine isoleucine (KD, 140 nM), by secretin (KD, 470 nM) and by the human pancreatic growth hormone-releasing factor (hpGRF; KD, 3200 nM). In the same acini preparation, 10 microM VIP also stimulated amylase release 4-fold and increased cyclic AMP 11-fold. Thus, VIP might be a neurotransmitter in the rat parotid gland.