Long-term survival of LGR5 expressing supporting cells after severe ototoxic trauma in the adult mouse cochlea.

Introduction: The leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a tissue resident stem cell marker, which it is expressed in supporting cells (SCs) in the organ of Corti in the mammalian inner ear. These LGR5+ SCs can be used as an endogenous source of progenitor cells for regeneration of hair cells (HCs) to treat hearing loss and deafness. We have recently reported that LGR5+ SCs survive 1 week after ototoxic trauma. Here, we evaluated Lgr5 expression in the adult cochlea and long-term survival of LGR5+ SCs following severe hearing loss. Methods: Lgr5GFP transgenic mice and wild type mice aged postnatal day 30 (P30) and P200 were used. P30 animals were deafened with a single dose of furosemide and kanamycin. Seven and 28 days after deafening, auditory brainstem responses (ABRs) were recorded. Cochleas were harvested to characterize mature HCs and LGR5+ SCs by immunofluorescence microscopy and quantitative reverse transcription PCR (q-RT-PCR). Results: There were no significant age-related changes in Lgr5 expression when comparing normal-hearing (NH) mice aged P200 with P30. Seven and 28 days after ototoxic trauma, there was severe outer HC loss and LGR5 was expressed in the third row of Deiters' cells and in inner pillar cells. Seven days after induction of ototoxic trauma there was an up-regulation of the mRNA expression of Lgr5 compared to the NH condition; 28 days after ototoxic trauma Lgr5 expression was similar to NH levels. Discussion: The presence of LGR5+ SCs in the adult mouse cochlea, which persists after severe HC loss, suggests potential regenerative capacity of endogenous cochlear progenitor cells in adulthood. To our knowledge, this is the first study showing not only long-term survival of LGR5+ SCs in the normal and ototoxically damaged cochlea, but also increased Lgr5 expression in the adult mouse cochlea after deafening, suggesting long-term availability of potential target cells for future regenerative therapies.

TH-MYCN tumors, but not tumor-derived cell lines, are adrenergic lineage, GD2+, and responsive to anti-GD2 antibody therapy.

Neuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma progression despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted. We demonstrate that neuroblasts in TH-MYCN-driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. This occurs in association with a transition from an adrenergic to mesenchymal differentiation state. Importantly, not only is GD2 expression retained on tumors in situ, treatment with a murine anti-GD2 antibody, 14G2a, markedly extends survival in such mice, including durable complete responses. Tumors in 14G2a-treated mice have fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironment. Our findings support the utility of this model to inform immunotherapy approaches for neuroblastoma and potential opportunities to investigate drivers of adrenergic to mesenchymal fate decisions.

[Epidemiology of fibrosing interstitial lung diseases in the department of Haute Garonne]. / EPIDemio : épidémiologie des pneumopathies interstitielles diffuses (PID) fibrosantes en Haute-Garonne.

EPIDemio study is a multicenter, prospective and observational study. The objective is to estimate the prevalence and incidence of fibrosing interstitial lung diseases (ILDs) in the department of Haute Garonne (31) in France. Fifty-five pulmonologists from the Toulouse university hospital and 8 private establishments participated in this study. Two hundred and fifty-six cases of fibrosing ILDs were reported (gross overall prevalence: 22.8/100,000 and estimated 30.1/100,000. Idiopathic ILDs represent 55.8% of fibrosing ILDs ahead of systemic disease-related ILDs (24.6%) and ILDs associated with environmental exposure (13.3%). Idiopathic pulmonary fibrosis (IPF) represents 35.9% of fibrosing ILDs, which corresponds to a minimal prevalence of 8.2/100,000 and an estimated prevalence of 11.2/100,000. This study confirms epidemiological data collected in France and Europe.

LGR5-Positive Supporting Cells Survive Ototoxic Trauma in the Adult Mouse Cochlea.

Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters' cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma.

Ouabain Does Not Induce Selective Spiral Ganglion Cell Degeneration in Guinea Pigs.

Round window membrane (RWM) application of ouabain is known to selectively destroy type I spiral ganglion cells (SGCs) in cochleas of several rodent species, while leaving hair cells intact. This protocol has been used in rats and Mongolian gerbils, but observations in the guinea pig are conflicting. This is why we reinvestigated the effect of ouabain on the guinea pig cochlea. Ouabain solutions of different concentrations were placed, in a piece of gelfoam, upon the RWM of the right cochleas. Auditory function was assessed using acoustically evoked auditory brainstem responses (aABR). Finally, cochleas were fixed and processed for histological examination. Due to variability within treatment groups, histological data was pooled and three categories based upon general histological observations were defined: cochleas without outer hair cell (OHC) and SGC loss (Category 1), cochleas with OHC loss only (Category 2), and cochleas with OHC and SGC loss (Category 3). Animals treated with 1 mM or 10 mM ouabain showed shifts in hearing thresholds, corresponding with varying histological changes in their cochleas. Most cochleas exhibited complete outer hair cell loss in the basal and middle turns, while some had no changes, together with either moderate or near-complete loss of SGCs. Neither loss of inner hair cells nor histological changes of the stria vascularis were observed in any of the animals. Cochleas in Category 1 had normal aABRs and morphology. On average, in Category 2 OHC loss was 46.0±5.7%, SGC loss was below threshold, ABR threshold shift was 44.9±2.7 dB, and ABR wave II amplitude was decreased by 17.1±3.8 dB. In Category 3 OHC loss was 68.3±6.9%, SGC loss was 49.4±4.3%, ABR threshold shift was 39.0±2.4 dB, and ABR amplitude was decreased by 15.8±1.6 dB. Our results show that ouabain does not solely destroy type I SGCs in the guinea pig cochlea.

Degeneration of auditory nerve fibers in guinea pigs with severe sensorineural hearing loss.

Damage to and loss of the organ of Corti leads to secondary degeneration of the spiral ganglion cell (SGC) somata of the auditory nerve. Extensively examined in animal models, this degeneration process of SGC somata following deafening is well known. However, degeneration of auditory nerve axons, which conduct auditory information towards the brainstem, and its relation to SGC soma degeneration are largely unknown. The consequences of degeneration of the axons are relevant for cochlear implantation, which is applied to a deafened system but depends on the condition of the auditory nerve. We investigated the time sequence of degeneration of myelinated type I axons in deafened guinea pigs. Auditory nerves in six normal-hearing and twelve deafened animals, two, six and fourteen weeks (for each group four) after deafening were histologically analyzed. We developed a semi-automated method for axon counting, which allowed for a relatively large sample size (20% of the total cross-sectional area of the auditory nerve). We observed a substantial loss of auditory nerve area (29%), reduction in axon number (59%) and decrease in axoplasm area (41%) fourteen weeks after deafening compared to normal-hearing controls. The correlation between axonal degeneration and that of the SGC somata in the same cochleas was high, although axonal structures appeared to persist longer than the somata, suggesting a slower degeneration process. In the first two weeks after induction of deafness, the axonal cross-sectional area decreased but the axon number did not. In conclusion, the data strongly suggest that each surviving SGC possesses an axon.

LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea.

In the developing cochlea, Wnt/ß-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/ß-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/ß-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/ß-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells (OHC). Supernumerary hair cells are likely formed due to an up-regulation of the "pro-hair cell" transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

Scar formation in mice deafened with kanamycin and furosemide.

In mammals, hair cell loss is irreversible and leads to hearing loss. To develop and test the functioning of different strategies aiming at hair cell regeneration, animal models of sensorineural hearing loss are essential. Although cochleae of these animals should lack hair cells, supporting cells should be preserved forming an environment for the regenerated hair cells. In this study, we investigated how ototoxic treatment with kanamycin and furosemide changes the structure of cochlear sensory epithelium in mice. The study also compared different tissue preparation protocols for scanning electron microscopy (SEM). Cochleae were collected from deafened and nondeafened mice and further processed for plastic mid modiolar sections and SEM. For comparing SEM protocols, cochleae from nondeafened mice were processed using three protocols: osmium-thiocarbohydrazide-osmium (OTO), tannic acid-arginine-osmium, and the conventional method with gold-coating. The OTO method demonstrated optimal cochlear tissue preservation. Histological investigation of cochleae of deafened mice revealed that the supporting cells enlarged and ultimately replaced the lost hair cells forming types 1 and 2 phalangeal scars in a base towards apex gradient. The type 3 epithelial scar, flattened epithelium, has not been seen in analysed cochleae. The study concluded that mice deafened with kanamycin and furosemide formed scars containing supporting cells, which renders this mouse model suitable for testing various hair cell regeneration approaches. Microsc. Res. Tech. 79:766-772, 2016. © 2016 Wiley Periodicals, Inc.

¹9F nuclear magnetic resonance screening of glucokinase activators.

Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R2 relaxation of the (19)F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the (19)F signal intensity after an 80-ms spin echo correlates nicely with the EC50, opening a route for NMR screening of GK activators.

Does vestibular end-organ function recover after gentamicin-induced trauma in Guinea pigs?

Until 1993 it was commonly accepted that regeneration of vestibular hair cells was not possible in mammals. Two histological studies then showed structural evidence for spontaneous regeneration of vestibular hair cells after gentamicin treatment. There is less evidence for functional recovery going along with this regenerative process; in other words, do regenerated hair cells function adequately? This study aims to address this question, and in general evaluates whether spontaneous functional recovery may occur, in the short or long term, in mammals after ototoxic insult. Guinea pigs were treated with gentamicin for 10 consecutive days at a daily dose of 125 mg/kg body weight. Survival times varied from 1 day to 16 weeks. Vestibular short-latency evoked potentials (VsEPs) to linear acceleration pulses were recorded longitudinally to assess otolith function. After the final functional measurements we performed immunofluorescence histology for hair cell counts. Auditory brainstem responses (ABRs) to click stimuli were recorded to assess cochlear function. As intended, gentamicin treatment resulted in significant loss of utricular hair cells and accompanying declines in VsEPs. Hair cell counts 8 or 16 weeks after treatment did not significantly differ from counts after shorter survival periods. Maximal functional loss was achieved 1-4 weeks after treatment. After this period, only 2 animals showed recovery of VsEP amplitude - all other animals did not reveal signs of regeneration or recovery. In contrast, after initial ABR threshold shifts there was a small but significant recovery. We conclude that spontaneous recovery of otolith function, in contrast to cochlear function, is very limited in guinea pigs. These results support the concept of intratympanic gentamicin treatment where gentamicin is used for chemoablation of the vestibular sensory epithelia.

Deafness induction in mice.

HYPOTHESIS: How to induce most efficiently severe sensorineural hearing loss in mice using a single coadministration of an aminoglycoside antibiotic and a loop diuretic? BACKGROUND: The coadministration of aminoglycosides and a loop diuretic has been widely used to induce hair cell and spiral ganglion cell loss in guinea pigs. However, the development of new treatment strategies against sensorineural hearing loss, such as tissue engineering techniques, requires the use of mouse models. Previous attempts to induce hearing loss in mice have rendered inconsistent results because of resistance to aminoglycoside-induced ototoxicity. Especially inner hair cells seem to be resistant to aminoglycoside-induced ototoxicity. METHODS: In the present study, we aim to optimize hearing loss in mice, using a single high-dose kanamycin (700 and 1,000 mg/kg) injection followed by a furosemide (100 mg/kg) administration. Although previous studies used intraperitoneal furosemide injections 30 minutes after kanamycin administration, we used intravenous furosemide injections administered within 5 minutes after kanamycin treatment. RESULTS: Auditory brain stem responses illustrated severe threshold shifts, and histologic analysis showed marked outer hair cell destruction as well as spiral ganglion cell loss. The present protocol results in more severe inner hair cell loss when compared with the results of previous researches. CONCLUSION: We conclude that severe sensorineural hearing loss can be induced in mice. Moreover, we found that this mouse model can be augmented via the use of rapid intravenous furosemide administrations to maximize inner hair cell loss.

The peripheral processes of spiral ganglion cells after intracochlear application of brain-derived neurotrophic factor in deafened guinea pigs.

OBJECTIVE: To characterize the effects of deafening and subsequent treatment with brain-derived neurotrophic factor (BDNF) on the peripheral processes (PPs) of spiral ganglion cells (SGCs) in guinea pigs. BACKGROUND: BDNF may prevent degeneration of neural structures after loss of hair cells with possible relevance for cochlear implant candidates. METHODS: Guinea pigs were deafened with a combination of kanamycin and furosemide. Two weeks after deafening, intracochlear BDNF treatment was started with osmotic pumps for 4 weeks. Two weeks after cessation of BDNF treatment, the cochleae were analyzed. PPs were counted and morphologically characterized with respect to myelination, size, and shape. RESULTS: Deafening reduced the number of PPs. We found that BDNF treatment, started 2 weeks after deafening, significantly reduced this degenerative effect. The remaining processes showed an altered morphology; compared with normal, the size was reduced in deafened untreated animals and increased in BDNF-treated animals. The myelin sheath seemed thinner in BDNF-treated animals. CONCLUSION: We conclude that BDNF has potential as an agent that can improve the interface between cochlear implants and the auditory periphery.

Covalent binding of 15-deoxy-delta12,14-prostaglandin J2 to PPARgamma.

Since 15-deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2)) has been identified as an endogenous ligand of PPARgamma thus inducing adipogenesis, it has been reported to play active parts in numerous cellular regulatory mechanisms. As 15dPGJ(2) has been shown to covalently bind several peptides and proteins, we investigated whether it also covalently binds PPARgamma. We first observed that after incubation of 15dPGJ(2) with recombinant PPARgamma, the quantity of free 15dPGJ(2) measured was always lower than the initial amount. We then measured the ability of the labeled agonist rosiglitazone to displace the complex PPARgamma(2)/15dPGJ(2) obtained after pre-incubation. We observed that the binding of rosiglitazone was dependent on the initial concentration of 15dPGJ(2). Finally using MALDI-TOF mass spectrometry analysis, after trypsinolysis of an incubate of the PPARgamma(2) ligand binding domain (GST-LBD) with 15dPGJ2, we found a fragment (m/z = 1314.699) corresponding to the addition of 15dPGJ(2) (m/z = 316.203) to the GST-LBD peptide (m/z = 998.481). All these observations demonstrate the existence of a covalent binding of 15dPGJ(2) to PPARgamma, which opens up new perspectives to study the molecular basis for selective activities of PPARs.

Potential involvement of adipocyte insulin resistance in obesity-associated up-regulation of adipocyte lysophospholipase D/autotaxin expression.

AIMS/HYPOTHESIS: Autotaxin is a lysophospholipase D that is secreted by adipocytes and whose expression is substantially up-regulated in obese, diabetic db/db mice. The aim of the present study was to depict the physiopathological and cellular mechanisms involved in regulation of adipocyte autotaxin expression. METHODS: Autotaxin mRNAs were quantified in adipose tissue from db/db mice (obese and highly diabetic type 2), gold-thioglucose-treated (GTG) mice (highly obese and moderately diabetic type 2), high-fat diet-fed (HFD) mice (obese and moderately diabetic type 2), streptozotocin-treated mice (thin and diabetic type 1), and massively obese humans with glucose intolerance. RESULTS: When compared to non-obese controls, autotaxin expression in db/db mice was significantly increased, but not in GTG, HFD, or streptozotocin-treated mice. During db/db mice development, up-regulation of autotaxin occurred only 3 weeks after the emergence of hyperinsulinaemia, and simultaneously with the emergence of hyperglycaaemia. Adipocytes from db/db mice exhibited a stronger impairment of insulin-stimulated glucose uptake than non-obese and HFD-induced obese mice. Autotaxin expression was up-regulated by treatment with TNFalpha (insulin resistance-promoting cytokine), and down-regulated by rosiglitazone treatment (insulin-sensitising compound) in 3T3F442A adipocytes. Finally, adipose tissue autotaxin expression was significantly up-regulated in patients exhibiting both insulin resistance and impaired glucose tolerance. CONCLUSIONS/INTERPRETATION: The present work demonstrates the existence of a db/db-specific up-regulation of adipocyte autotaxin expression, which could be related to the severe type 2 diabetes phenotype and adipocyte insulin resistance, rather than excess adiposity in itself. It also showed that type 2 diabetes in humans is also associated with up-regulation of adipocyte autotaxin expression.

Design, synthesis and in vitro evaluation of novel benzo[b]thiophene derivatives as serotonin N-acetyltransferase (AANAT) inhibitors.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is the penultimate enzyme in melatonin (5-methoxy-N-acetyltryptamine) biosynthesis. It is the key-enzyme responsible of the nocturnal rhythm of melatonin production in the pineal gland. Specific AANAT inhibitors could be useful for treatment of different physiopathological disorders encountered in diseases such as seasonal affective disorders or obesity. On the basis of previous works and 3D-QSAR studies carried out in our laboratory, we have synthesized and evaluated four novel benzo[b]thiophene derivatives designed as AANAT inhibitors. Compound 13 exhibited high inhibitory activity (IC50 = 1.4 microM) and low affinities for both MT, (1100 nM) and MT2 (1400 nM) receptors.

Characterization and regulation of a CHO cell line stably expressing human serotonin N-acetyltransferase (EC 2.3.1.87).

Current melatonin research is essentially based on the finding of new molecular tools, including synthetic or natural agonists and antagonists for the melatonin receptors and synthetic inhibitors of the enzymes involved in its biosynthesis. Indeed, the use of these compounds will improve our understanding of some of the numerous mechanisms of action of melatonin. The present report deals with the establishment and description of a new cell line expressing in a stable manner human arylalkylamine-N-acetyltransferase (AANAT, E.C.2.3.1.87). This new cellular system permits one to check the capacity of newly discovered inhibitors to penetrate the cell and reach their target. Some emphasis is put on inhibitors of the bromoacetyltryptamine family since these precursor compounds form in situ bisubstrate inhibitors with strong affinity for the human enzyme. AANAT is known to undergo complex and rapid regulation by a subtle balance between extremely fast catabolism and protection against it, both due to serine phosphorylation. In the present report, this phosphorylation is shown to occur in vitro after incubation with several kinases (rho-kinase, chk-1, protein kinase A) but not with protein kinase C. Phosphorylation enhances the specific activity of the enzyme by a factor of two to five. This phosphorylation is also shown to occur after treatment of the cell with compounds such as forskolin and rolipram that enhance or protect the intracellular pool of cAMP or the cell-permeable cAMP analogue, dioctanoyl-cAMP. The specificity of the cellular model was assessed using a series of substrates and inhibitors of AANAT already described in the literature, and the characteristics of this cellular system are shown to correspond with those reported for the purified enzyme. This cell line was used to screen libraries of compounds in a living system and led to the discovery of several potent specific and non-toxic AANAT inhibitors.

Design, synthesis and in vitro evaluation of novel derivatives as serotonin N-acetyltransferase inhibitors.

Serotonin N-acetyltransferase (arylalkylamine N-acetyl-transferase, AANAT) is an enzyme that catalyses the first rate limiting step in the biosynthesis of melatonin (5-methoxy-N-acetyltryptamine). Different physiopathological disorders in human may be due to abnormal secretion of melatonin leading to an inappropriate exposure of melatonin receptors to melatonin. For that reason, we have designed, synthesized and evaluated as inhibitors of human serotonin N-acetyltransferase, a series of compounds that were able to react with coenzyme A to give a bisubstrate analog inhibitor. Compound 12d was found to be a potent AANAT inhibitor (IC50 = 0.18 microM).

Development of new assays and improved procedures for the purification of recombinant human chymase.

Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.

Functional evidence for a role of vascular chymase in the production of angiotensin II in isolated human arteries.

BACKGROUND: In human arteries, angiotensin-converting enzyme (ACE) inhibitors incompletely block the production of angiotensin (Ang) II from Ang I. This ACE-independent production of Ang II appears to be caused by serine proteases, one of which presumably is chymase. However, several serine proteases may produce Ang II, and the exact role of chymase in the vascular production of Ang II has never been directly evaluated using selective chymase inhibitors. METHODS AND RESULTS: Rings of human mammary arteries were subjected to either Ang I or the chymase-selective substrate [pro,(11) D-Ala(12)] Ang I in the absence or the presence of the ACE inhibitor captopril, the serine protease inhibitor chymostatin, or the selective chymase inhibitor C41. Captopril only partially inhibited (by 33%) the response to Ang I. In the absence of captopril, C41 markedly reduced (by 44%) the response to Ang I, and this effect was identical to that of chymostatin. C41 also significantly reduced the response to Ang I in the presence of captopril, although this inhibitory effect was slightly less than that of captopril in combination with chymostatin. [Pro,(11)D-Ala(12)] Ang I induced potent contractions that were not affected by captopril but were abolished by chymostatin and markedly reduced by C41. In addition, we found that prior treatment of the patients with an ACE inhibitor did not affect the in vitro response to Ang I (in the absence or the presence of captopril) or to [Pro,(11)D-Ala(12)] Ang I. CONCLUSIONS: Our results reinforce the hypothesis that chymase is a major serine protease implicated in the ACE-independent production of Ang II in human arteries.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN --> RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K(m) for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.