Inverse agonism and neutral antagonism at wild-type and constitutively active mutant delta opioid receptors.

The delta opioid receptor modulates nociceptive and emotional behaviors. This receptor has been shown to exhibit measurable spontaneous activity. Progress in understanding the biological relevance of this activity has been slow, partly due to limited characterization of compounds with intrinsic negative activity. Here, we have used constitutively active mutant (CAM) delta receptors in two different functional assays, guanosine 5'-O-(3-thio)triphosphate binding and a reporter gene assay, to test potential inverse agonism of 15 delta opioid compounds, originally described as antagonists. These include the classical antagonists naloxone, naltrindole, 7-benzylidene-naltrexone, and naltriben, a new set of naltrindole derivatives, H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-TicPsi[CH2N]Cha-Phe-OH [TICP(Psi)], as well as three 2',6'-dimethyltyrosine-1,2,3,4-tetrahydroquinoline-3-carboxylate (Dmt-Tic) peptides. A reference agonist, SNC 80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], and inverse agonist, ICI 174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu), were also included. In a screen using wild-type and CAM M262T delta receptors, naltrindole (NTI) and close derivatives were mostly inactive, and TIPP behaved as an agonist, whereas Dmt-Tic-OH and N,N(CH3)2-Dmt-Tic-NH2 showed inverse agonism. The two latter compounds showed negative activity across 27 CAM receptors, suggesting that this activity was independent from the activation mechanism. These two compounds also exhibited nanomolar potencies in dose-response experiments performed on wild-type, M262T, Y308H, and C328R CAM receptors. TICP(Psi) exhibited strong inverse agonism at the Y308H receptor. We conclude that the stable N,N(CH3)2-Dmt-Tic-NH2 compound represents a useful tool to explore the spontaneous activity of delta receptors, and NTI and novel derivatives behave as neutral antagonists.

A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling.

Large scale sequencing of the human mu-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (beta-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, beta-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human mu-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo.

[Impact of initial antibiotic therapy on the course of resistance to fluoroquinolones and aminoglycosides in Gram-negative bacilli isolated from intensive care patients]. / Impact de l'antibiothérapie initiale sur l'évolution des résistances aux fluoroquinolones et aux aminosides des bacilles à gram négatif isolés chez des patients de réanimation.

OBJECTIVE: To evaluate the effect of the initial antibiotic therapy associating a betalactam antibiotic (BLA) with either an aminoglycoside (AG) or a fluoroquinolone (FQ) on the development of resistance of gram-negative bacilli in an intensive care unit. STUDY DESIGN: Prospective bacteriological surveillance study. PATIENTS: The study included 51 patients experiencing a second infection with gram-negative organisms, eight days or more after a first infection. METHOD: The incidences of bacterial infection and the antimicrobial susceptibility have been assessed. RESULTS: The first-choice therapy was based either on BLA + AG (51%), or on BLA + FQ in the others (46%). The causative organisms were Enterobacteriaceae (57%) and Pseudomonas aeruginosa (31%). The second infection occurred 23 +/- 11 days after the first. The main organisms involved were Pseudomonas aeruginosa (51%) and Enterobacteriaceae (41%). In the group treated initially with an AG, only the antibiotic susceptibility for amikacin decreased significantly (72 vs 36%, p < 0.05). The latter was the most prescribed antibiotic (56%). In the FQ group, there was a significant decrease of susceptibility for ciprofloxacin, pefloxacin, netilmicin and tobramycin. The decrease was not significant for gentamicin and amikacin. CONCLUSIONS: In intensive care patients, the use of FQ in association with a BLA increases the resistance to AG and FQ. Therefore it seems preferable to administer an AG in association with a BLA. Amikacine should only be prescribed when justified for a given case.

Haemodynamic changes induced by two I:E ratios: a transoesophageal echocardiographic study.

PURPOSE: To assess the effects of controlled ventilation with two I:E ratios on haemodynamic and left ventricular function in mechanically ventilated patients with moderate to severe respiratory disease, using fluctuation of the arterial pressure waveform and the changes in left ventricular areas obtained by transoesophageal echocardiography. METHODS: Nine patients had their lungs ventilated using volume controlled ventilation with two I:E ratios 1:3 and 1:1). Respiratory rate was adjusted so that six cardiac beats occurred during a respiratory cycle. Systolic blood pressure variation (SBPV), left ventricular area variations measured by TEE and haemodynamic variables measured by PA catheter were compared. RESULTS: When compared with I:E (1:3), I:E (1:1) decreased end diastolic area (EDA) throughout the respiratory cycle from 3% to 8% (P < 0.01) and increased SBPV from 6 +/- 1 to 11 +/- 1 mmHg (P < 0.01). In four patients, SBPV was > 12 mmHg with I:E 1:1. Conversely, SBPV was < 10 mmHg in all patients with I:E 1:3. With I:E (1:1), EDA decreased up to 7% during expiration (P < 0.01). The ejection fraction area remained stable for both ventilatory patterns and throughout the ventilatory cycle for a given I:E. The usual invasive haemodynamic variables were unchanged throughout the study, as was PaO2/FIO2. CONCLUSION: In this setting, EDA and SBPV allow beat-to-beat evaluation of left ventricular preload during change of I:E ratio. Switch from I:E 1:3 to 1:1 may be used as a rapid, safe and reversible test to estimate intravascular volume status assessed by changes in SBPV or EDA.