Emerging resistance mutations in PI-naive patients failing an atazanavir-based regimen (ANRS multicentre observational study).

Background: Atazanavir is a PI widely used as a third agent in combination ART. We aimed to determine the prevalence and the patterns of resistance in PI-naive patients failing on an atazanavir-based regimen. Methods: We analysed patients failing on an atazanavir-containing regimen used as a first line of PI therapy. We compared the sequences of reverse transcriptase and protease before the introduction of atazanavir and at failure [two consecutive viral loads (VLs) >50 copies/mL]. Resistance was defined according to the 2014 Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) algorithm. Results: Among the 113 patients, atazanavir was used in the first regimen in 71 (62.8%) patients and in the first line of a PI-based regimen in 42 (37.2%). Atazanavir was boosted with ritonavir in 95 (84.1%) patients and combined with tenofovir/emtricitabine or lamivudine (n = 81) and abacavir/lamivudine or emtricitabine (n = 22). At failure, median VL was 3.05 log10 copies/mL and the median CD4+ T cell count was 436 cells/mm3. The median time on atazanavir was 21.2 months. At failure, viruses were considered resistant to atazanavir in four patients (3.5%) with the selection of the following major atazanavir-associated mutations: I50L (n = 1), I84V (n = 2) and N88S (n = 1). Other emergent PI mutations were L10V, G16E, K20I/R, L33F, M36I/L, M46I/L, G48V, F53L, I54L, D60E, I62V, A71T/V, V82I/T, L90M and I93L/M. Emergent NRTI substitutions were detected in 21 patients: M41L (n = 2), D67N (n = 3), K70R (n = 1), L74I/V (n = 3), M184V/I (n = 16), L210W (n = 1), T215Y/F (n = 3) and K219Q/E (n = 2). Conclusions: Resistance to atazanavir is rare in patients failing the first line of an atazanavir-based regimen according to the ANRS. Emergent NRTI resistance-associated mutations were reported in 18% of patients.

Prevalence of HIV-1 drug resistance in treated patients with viral load >50 copies/mL: a 2014 French nationwide study.

Background: Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. Methods: The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. Results: The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. Conclusion: In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.

Gene transfer of two entry inhibitors protects CD4⁺ T cell from HIV-1 infection in humanized mice.

Targeting viral entry is the most likely gene therapy strategy to succeed in protecting the immune system from pathogenic HIV-1 infection. Here, we evaluated the efficacy of a gene transfer lentiviral vector expressing a combination of viral entry inhibitors, the C46 peptide (an inhibitor of viral fusion) and the P2-CCL5 intrakine (a modulator of CCR5 expression), to prevent CD4⁺ T-cell infection in vivo. For this, we used two different models of HIV-1-infected mice, one in which ex vivo genetically modified human T cells were grafted into immunodeficient NOD.SCID.γc⁻/⁻mice before infection and one in which genetically modified T cells were derived from CD34⁺ hematopoietic progenitors grafted few days after birth. Expression of the transgenes conferred a major selective advantage to genetically modified CD4⁺ T cells, the frequency of which could increase from 10 to 90% in the blood following HIV-1 infection. Moreover, these cells resisted HIV-1-induced depletion, contrary to non-modified cells that were depleted in the same mice. Finally, we report lower normalized viral loads in mice having received genetically modified progenitors. Altogether, our study documents that targeting viral entry in vivo is a promising avenue for the future of HIV-1 gene therapy in humans.

Pharmacokinetics of dolutegravir in a premature neonate after HIV treatment intensification during pregnancy.

We describe the pharmacokinetics of dolutegravir (DTG) in a premature neonate after maternal intensification of an antiretroviral (ARV) regimen by adding DTG. During the last 2 weeks of pregnancy, the ARV was tenofovir-emtricitabine, atazanavir-ritonavir, and DTG (50 mg once daily). From the interaction between atazanavir and DTG via CYP3A4 and UGT1A1 and placental efflux transporter inhibition and considering the infant's probable enzymatic immaturity, the DTG elimination half-life was estimated to be 4-fold longer in neonates than in adults.

Reasons for attending an urban urgent care centre with minor illness: a questionnaire study.

INTRODUCTION: The demand for urgent care is increasing, and the pressure on emergency departments is of significant concern. General practitioner (GP)-led urgent care centres are a new model of care developed to divert patients to more appropriate primary care environments. This study explores why patients with minor illness choose to attend an urban urgent care centre for their healthcare needs. METHODS: A self-completed questionnaire among patients aged 18 years or over (N=649) who were triaged with a 'minor illness' on arrival to an urgent care centre, colocated with an emergency department in London. RESULTS: Median participant age was 29 years. 58% (649/1112) of patients attending the centre with minor illness during the study period took part. 72% participants were registered with a GP; more women (59%) attended than men; and the majority of participants rated themselves as healthy (81%). Access to care (58%) was a key reason for using the service as was expectation of receiving prescription medication (69%). GP dissatisfaction influenced 10% of participants in their decision to attend. 68% did not contact their GP in the previous 24 h before attending. CONCLUSIONS: We found that the GP-led urgent care centre was similar to walk in centres in attracting healthy young adults, who were mostly registered with a GP and used services because of convenience and ease of access rather than satisfaction levels with their GP. This group may benefit from being seen as part of routine general practice care to provide opportunities for education and promotion of self-management.

Long-term follow-up of 11 protease inhibitor (PI)-naïve and PI-treated HIV-infected patients harbouring virus with insertions in the HIV-1 protease gene.

OBJECTIVES: Amino acid insertions in the protease gene have been reported rarely, and mainly in patients receiving protease inhibitors (PIs). The aim of the study was to assess the long-term viro-immunological follow-up of HIV-infected patients harbouring virus with protease insertions. METHODS: Cases of virus exhibiting protease insertions were identified in routine resistance genotyping tests. Therapeutic, immunological and virological data were retrospectively collected. RESULTS: Eleven patients harbouring virus with a protease gene insertion were detected (prevalence 0.24%), including three PI-naïve patients. The insertions were mainly located between codons 33 and 39 and associated with surrounding mutations (M36I/L and R41K). The three PI-naïve patients were infected with an HIV-1 non-B subtype. Follow-up of these PI-naïve patients showed that the insert-containing virus persisted for several years, was archived in HIV DNA, and displayed a reduced viral replicative capacity with no impact on resistance level. Of the eight PI-experienced patients, 63% were infected with HIV-1 subtype B; one had been antiretroviral-free for 5 years and seven were heavily PI-experienced (median duration of follow-up 24 months; range 10-62 months). The protease insertion was selected under lopinavir in four patients and under darunavir in one, in the context of major PI-resistance mutations, and following long-term exposure to PIs. The insert-containing virus persisted for a median of 32 months (range 12-62 months) and displayed no specific impact on phenotypic resistance level or viral replicative capacity. CONCLUSION: Our data, obtained during long-term follow-up, show that insertions in the protease gene do not seem to have an impact on resistance level. This finding supports the recommendation of PI-based regimens, although further work is required to confirm it.

Laponite/poly(2-methyl-2-oxazoline) hydrogels: Interplay between local structure and rheological behaviour.

HYPOTHESIS: Dispersions of Laponite in water may form gels, the rheological properties of which being possibly tuned by the addition of polymer chains. Laponite-based hydrogels with poly(ethylene oxide) (PEO) were the most widely investigated systems and the PEO chains were then found to reduce the elastic modulus. EXPERIMENTS: Here, hydrogels based on Laponite and poly(2-methyl-2-oxazoline) (POXA) were considered. The adsorption behavior and the local structures within these nanocomposite gels were investigated by small-angle neutron scattering and NMR. The same materials were macroscopically characterized using rheology. FINDINGS: An original evolution of the storage modulus G' with the POXA concentration is evidenced compared to Laponite/PEO hydrogels. At low POXA concentrations, a continuous reduction of G' is observed upon increasing the polymer content, as with PEO, due to the screening of electrostatic interactions between the clay platelets. However, above a critical value of the POXA concentration, G' increases with the polymer content. This difference with PEO-based hydrogels is correlated to the stronger affinity of POXA chains for the clay surfaces, which results in the reduction of the inhomogeneities for the Laponite disks within the gels. Steric repulsions would then counterbalance the effect of electrostatic repulsions and lead to the strengthening of the POXA-based hydrogels.

Daily multidisciplinary COVID-19 meeting: Experiences from a French university hospital.

OBJECTIVES: In March 2020, the World Health Organization declared the coronavirus disease 2019 (COVID-19) a pandemic. In absence of official recommendations, implementing daily multidisciplinary team (MDT) COVID-19 meetings was urgently needed. Our aim was to describe our initial institutional standard operating procedures for implementing these meetings, and their impact on daily practice. METHODS: All consecutive patients who were hospitalized in our institution due to COVID 19, from March 31 to April 15, 2020, were included. Criteria to be presented at MDT meetings were defined as a proven COVID-19 by PCR or strongly suspected on CT scan, requiring hospitalization and treatment not included in the standard of care. Three investigators identified the patients who met the predefined criteria and compared the treatment and outcomes of patients with predefined criteria that were presented during MDT meeting with those not presented during MDT meeting. COVID-19 MDT meeting implementation and adhesion were also assessed by a hospital medical staff survey. RESULTS: In all, 318 patients with confirmed or suspected COVID-19 were examined in our hospital. Of these, 230 (87%) were hospitalized in a COVID-19 unit, 91 (40%) of whom met predefined MDT meeting criteria. Fifty (55%) patients were presented at a MDT meeting versus 41 (45%) were not. Complementary exploration and inclusion in the CorImmuno cohort were higher in MDT meeting group (respectively 35 vs. 15%, P=0.03 and 80 versus 49%, P=0.0007). Prescription of hydrocortisone hemisuccinate was higher in group of patients not presented during MDT meeting (24 vs. 51%, P=0.007). Almost half of the patients fulfilling the inclusion criteria were not presented at MDT meeting, which can be partly explained by technical software issues. CONCLUSIONS: Multidisciplinary COVID-19 meetings helped implementing a single standard of care, avoided using treatments that were untested or currently being tested, and facilitated the inclusion of patients in prospective cohorts and therapeutic trials.

Biodiversity analysis by polyphasic study of marine bacteria associated with biocorrosion phenomena.

AIMS: A polyphasic approach was used to study the biodiversity bacteria associated with biocorrosion processes, in particular sulfate-reducing bacteria (SRB) and thiosulfate-reducing bacteria (TRB) which are described to be particularly aggressive towards metallic materials, notably via hydrogen sulfide release. METHODS AND RESULTS: To study this particular flora, an infrared spectra library of 22 SRB and TRB collection strains were created using a Common Minimum Medium (CMM) developed during this study and standardized culture conditions. The CMM proved its ability to allow for growth of both SRB and TRB strains. These sulfurogen collection strains were clearly discriminated and differentiated at the genus level by fourier transform infrared (FT-IR) spectroscopy. In a second step, infrared spectra of isolates, recovered from biofilms formed on carbon steel coupons immersed for 1 year in three different French harbour areas, were compared to the infrared reference spectra library. In parallel, molecular methods (M13-PCR and 16S rRNA gene sequencing) were used to qualitatively evaluate the intra- and inter-species genetic diversity of biofilm isolates. The biodiversity study indicated that strains belonging to the Vibrio genus were the dominant population; strains belonging to the Desulfovibrio genus (SRB) and Peptostreptococcaceae were also identified. CONCLUSION: Overall, the combination of the FT-IR spectroscopy and molecular approaches allowed for the taxonomic and ecological study of a bacterial flora, cultivated on CMM, associated with microbiology-induced corrosion (MIC) processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Via the use of the CMM medium, the culture of marine bacteria (including both SRB and TRB bacteria) was allowed, and the implication of nonsulforogen bacteria in MIC was observed. Their involvement in the biocorrosion phenomena will have to be studied and taken into account in the future.

Performance of genotypic algorithms for predicting tropism for HIV-1 CRF01_AE recombinant.

OBJECTIVES: There is no consensus about the performances of genotypic rules for predicting HIV-1 non-B subtype tropism. Three genotypic methods were compared for CRF01_AE HIV-1 tropism determination. METHODS: The V3 env region of 207 HIV-1 CRF01_AE and 178 B subtypes from 17 centers in France and 1 center in Switzerland was sequenced. Tropism was determined by Geno2Pheno algorithm with false positive rate (FPR) 5% or 10%, the 11/25 rule or the combined criteria of the 11/25, net charge rule and NXT/S mutations. RESULTS: Overall, 72.5%, 59.4%, 86.0%, 90.8% of the 207 HIV-1 CRF01_AE were R5-tropic viruses determined by Geno2pheno FPR5%, Geno2pheno FPR10%, the combined criteria and the 11/25 rule, respectively. A concordance of 82.6% was observed between Geno2pheno FPR5% and the combined criteria for CRF01_AE. The results were nearly similar for the comparison between Geno2pheno FPR5% and the 11/25 rule. More mismatches were observed when Geno2pheno was used with the FPR10%. Neither HIV viral load, nor current or nadir CD4 was associated with the discordance rate between the different algorithms. CONCLUSION: Geno2pheno predicted more X4-tropic viruses for this set of CRF01_AE sequences than the combined criteria or the 11/25 rule alone. For a conservative approach, Geno2pheno FPR5% seems to be a good compromise to predict CRF01_AE tropism.

Mechanisms whereby extracellular adenosine 3',5'-monophosphate inhibits phosphate transport in cultured opossum kidney cells and in rat kidney. Physiological implication.

The mechanism of phosphaturia induced by cAMP infusion and the physiological role of extracellular cAMP in modulation of renal phosphate transport were examined. In cultured opossum kidney cells, extracellular cAMP (10-1,000 microM) inhibited Na-dependent phosphate uptake in a time- and concentration-dependent manner. The effect of cAMP was reproduced by ATP, AMP, and adenosine, and was blunted by phosphodiesterase inhibitors or by dipyridamole which inhibits adenosine uptake. [3H]cAMP was degraded extracellularly into AMP and adenosine, and radioactivity accumulated in the cells as labeled adenosine and, subsequently, as adenine nucleotides including cAMP. Radioactivity accumulation was decreased by dipyridamole and by inhibitors of phosphodiesterases and ecto-5'-nucleotidase, assessing the existence of stepwise hydrolysis of extracellular cAMP and intracellular processing of taken up adenosine. In vivo, dipyridamole abolished the phosphaturia induced by exogenous cAMP infusion in acutely parathyroidectomized (APTX) rats, decreased phosphate excretion in intact rats, and blunted phosphaturia induced by PTH infusion in APTX rats. These results indicate that luminal degradation of cAMP into adenosine, followed by cellular uptake of the nucleoside by tubular cells, is a key event which accounts for the phosphaturic effect of exogenous cAMP and for the part of the phosphaturic effect of PTH which is mediated by cAMP added to the tubular lumen under the influence of the hormone.

Evidence for a parathyroid hormone-independent calcium modulation of phosphate transport along the nephron.

To disclose a parathyroid-independent calcium modulation of phosphate transport along the nephron, the effect of increasing plasma calcium concentration to subnormal levels in rats 6 days after parathyroidectomy (chronic PTX) was studied. Fractional phosphate reabsorption was significantly increased. The whole kidney response to calcium infusion was similar whether or not the thyroid gland was removed, which suggests that calcitonin is not involved. The micropuncture study indicated an increase in the reabsorptive capacity for phosphate (absolute reabsorption/absolute delivered phosphate per nephron segment) in the proximal tubule, the loop, and the terminal nephron when calcium was infused. Thus, the level of plasma calcium or some related factor affects the phosphate transport by the tubule independently of parathyroid hormone. With calcium infusion, the profile of phosphate reabsorption along the nephron became close to that of acutely parathyroidectomized rats, but with persisting differences. The level of plasma calcium concentration may partly account for the differences between the acute and the chronic steps of parathyroidectomy. The role of possible interferences between alterations of extracellular calcium concentration or some related factor and the adenylate cyclase-cyclic AMP system in such an action of calcium was evaluated. Cyclic AMP was infused so as to achieve a 10(-6) M plasma concentration. Combined infusions of calcium and cyclic AMP were also performed. The results are compatible with calcium inhibition of adenylate cyclase, although they do not rule out a direct action of calcium.

Photoresponsive viscosity and host-guest association in aqueous mixtures of poly-cyclodextrin with azobenzene-modified poly(acrylic)acid.

In aqueous solutions, beta-cyclodextrin (CD) and cyclodextrin-containing polymers (PolCD) associate with azobenzene-modified polyacrylate (AMP). Inclusion complexes in solution of CD (or PolCD) and AMP, and the viscosity of these mixtures, have been studied as a function of the composition of AMP and concentrations of samples. AMPs are random copolymers containing a low fraction of a light-responsive hydrophobic moieties (<10 mol % of 6-[4-alkylamido]phenylazobenzene acrylamide), and a charged hydrophilic unit, sodium acrylate. PolCDs are beta-cyclodextrin randomly conjugated with epichlorohydrin and fractionated to yield copolymers of average number of CD per chain equal to 50. In dilute solutions, the composition of complexes has been investigated by capillary electrophoresis and UV-vis spectrometry. Association between PolCD and AMP appears more complex than the conventional Benesi-Hildebrand scheme. We identified a tight (quantitative) binding regime followed by a gradual increase of the density of AMP-bound PolCD upon increasing the concentration of PolCD. At higher concentrations, the formation of large clusters has been characterized by the increase of viscosity by several decades. Light-triggered trans-conformation of the azobenzene moieties of AMPs leads to a marked photoswitch of viscosity. Reversible viscosity swings by up to 6-fold were achieved by alternative exposure to UV and visible lights. In contrast, the composition of PolCD/AMP complexes in dilute regime does not respond to light, though subtle modifications of the structures of complexes are reflected by variation of electrophoretic mobilities and UV spectra. The properties of interpolymer clusters and photoviscosity are accordingly the result of modification of the dynamics of association. In practice, the low concentration of photochrome makes it possible to obtain rapid responses in samples having a thickness of the order of cm. The data reported provide guidelines for the formulations of CD/polymer systems, specifically, viscosity enhancers, which should show promising developments in pharmaceuticals or cosmetics.

Spontaneous association of hydrophobized dextran and poly-beta-cyclodextrin into nanoassemblies. Formation and interaction with a hydrophobic drug.

New nanoassemblies were instantaneously prepared by mixing two aqueous solutions, one containing a beta-cyclodextrin polymer (pbetaCD), and the other a hydrophobically modified by alkyl chains dextran (MD). The formation mechanism and the inner structure of these nanoassemblies were analysed using surface tension measurements and (1)H NMR spectroscopy. The effect of a hydrophobic guest molecule, such as benzophenone (BZ), on the formation and stability of the nanoassemblies was also evaluated. MD exhibited the typical behaviour of a soluble amphiphilic molecule and adsorbed at the air/water interface. Whereas the injection of native beta-CDs in the solution beneath the adsorbed MD monolayer did not produce any change in the surface tension, that of the pbetaCD resulted in an increase in the surface tension, indicating the desorption of the polymer from the interface. This result accounts for a cooperative effect of beta-CDs linked together in the pbetaCD polymer on dextran desorption. The presence of benzophenone in the system hindered the sequestration of dextran alkyl moieties by beta-CD in the polymer without impeding the formation of associative nanoassemblies of 100-200 nm. (1)H NMR investigations demonstrated that, in the BZ-loaded nanoassemblies, the hydrophobic molecule was mainly located into the cyclodextrin cavities.

Usefulness of FTIR spectroscopy to distinguish rough and smooth variants of Lactobacillus farciminis CNCM-I-3699.

In this study, the potential of Fourier transform infrared (FTIR) spectroscopy for assessing putative biochemical and structural differences between the two variants, rough (R) and smooth (S), of Lactobacillus farciminis CNCM-I-3699, a pleomorphic strain, was investigated. The main differences observed were localized in the polysaccharide (1200-900 cm-1) and protein (1700-1500 cm-1) regions. Based on spectral information in these two spectral ranges, clustering resulted in a dendrogram that showed a clear discrimination between both morphotypes. Significant increases in favor of morphotype S compared to R at specific wavenumbers for polysaccharides (22.18% vs. 5.24% at 1068 cm-1) and capsular polysaccharides (16% vs. 13.17% at 1048 cm-1) were recorded. Compared to S, the morphotype R exhibits a 1.27-fold higher signal at the wavenumber of 1637 cm-1 assigned to the amide I ß-sheet and a 2.71-fold higher signal at the wavenumber of 1513 cm-1 assigned to the tyrosine involved in the ß-sheet arrangement of proteins. The FTIR analysis is efficient to separate and give data on mainly surface component differences observed previously between S colony morphotype (ropy and exopolysaccharide positive) and the R colony morphotype (non-ropy but highly autoaggregative).

[Adherence to antiretroviral therapy during HIV infection, a multidisciplinary approach]. / "L'observance thérapeutique au cours de l'infection VIH, une approche multidisciplinaire".

Since HIV infection has become a chronic disease, antiretroviral therapy is now used on a long-term basis. Response to treatment is conditioned by numerous inter-dependent factors, including non-compliance, which can result in failure of the therapeutic regimen. Although compliance is crucial for long-term efficacy of the treatment, it is a dynamic factor, and therefore difficult to evaluate. This literature review proposes a multidisciplinary approach to treatment adherence during HIV infection, and deals with the following questions: how should adherence and non-adherence be defined? How are they correlated to the treatment response? How is adherence measured in trials and cohorts, as well as in clinical practice? By what factors is it influenced? What tools can be implemented to improve adherence? The interaction between adherence and response to antiretroviral therapy requires communication between clinicians, healthcare providers, patients, virologists, pharmacologists, and the companies responsible for developing drugs. The pharmaceutical industry must sustain its efforts to ensure a balance between demands for efficacy and adherence when developing new drugs. And the methods implemented by numerous healthcare teams plead in favour of a dynamic approach to adherence, with the active participation of all.

Protein kinase C activators and bradykinin selectively inhibit vasopressin-stimulated cAMP synthesis in MDCK cells.

To evaluate a possible modulation by protein kinase C of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of protein kinase C activators and of bradykinin on intracellular cAMP accumulation in MDCK cells. A 15-min pretreatment of cells with phorbol 12-myristate 13-acetate or 1-oleoyl-2-acetylglycerol induced a dose-dependent inhibition of vasopressin-stimulated cAMP synthesis, but not of basal or glucagon-, prostaglandin E2-, and forskolin-stimulated cAMP generation. 4 alpha-Phorbol 12,13-didecanoate, inactive on protein kinase C, did not affect cAMP accumulation. Bradykinin (0.1-10 microM) also inhibited the stimulatory effect of vasopressin on cAMP synthesis in a concentration-dependent manner, but affected neither basal cAMP content, nor its stimulation by glucagon, prostaglandin E2 and forskolin. The effect of activators of protein kinase C and of bradykinin occurred while renal prostaglandin synthesis was blocked with indomethacin. The inhibitory effect of protein kinase C activators and bradykinin on cAMP generation was reversed by the protein kinase C inhibitor H7, was enhanced by monensin, one effect of which is to block the recycling of membrane receptors, and persisted when the GTP-binding protein N1 was blocked with 1 mM Mn2+. Our data suggest that: protein kinase C can modulate the tubular effects of vasopressin by inhibiting cAMP generation; this effect is not mediated by renal prostaglandins, and might result from a direct action on the vasopressin receptor, or on its coupling with Ns; the modulation by bradykinin of vasopressin effects are likely to be exerted, at least partly, through activation of protein kinase C.

Increase in membrane fluidity modulates sodium-coupled uptakes and cyclic AMP synthesis by renal proximal tubular cells in primary culture.

In order to evaluate the influence of membrane fluidization on three apical transport systems and on a basolateral enzyme, and to analyse the mechanisms involved, we studied, in cultured rabbit proximal tubular cells, the effect of increasing concentrations of the local anesthetic drug benzyl alcohol on Na(+)-dependent uptakes of phosphate (Pi), methyl alpha-D-glucopyranoside (MGP), and L-alanine, as well as on basal and stimulated cyclic AMP content. At 10 mM, benzyl alcohol increased the Vmax of Pi uptake by 31%, decreased that of MGP uptake by 24%, and did not affect alanine uptake. Km values were not affected. Benzyl alcohol, up to 40 mM, increased in a concentration-dependent manner basal, PTH-stimulated, and cholera toxin-stimulated, but not forskolin-stimulated cyclic AMP accumulation. In the presence of 40 mM benzyl alcohol, the magnitude of PTH-induced inhibition of Pi uptake was enhanced from 11% to 24%. It is concluded that: (i) fluidization of apical membranes affected differently Na+/Pi, Na+/MGP, and Na+/alanine cotransports, reflecting differences in the lipidic environments of these transport system; (ii) fluidization of basolateral membranes enhanced PTH-stimulated cyclic AMP generation through improved coupling between the receptor-GS complex and the catalytic subunit of adenylate cyclase; (iii) these variations may result in physiological and pathophysiological modulation of the renal handling of solutes and of the phosphaturic effect of PTH.