Serum Nostrin-A risk factor of death, kidney replacement therapy and acute kidney disease in acute kidney injury.

BACKGROUND: The prediction of Acute Kidney Injury (AKI)-related outcomes remains challenging. Persistent kidney excretory dysfunction for longer than 7 days has been defined as Acute Kidney Disease (AKD). In this study, we prospectively quantified serum Nostrin, an essential regulator of endothelial NO metabolism, in hospitalized patients with AKI. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In-hospital subjects with AKI of various etiology were identified through the in-hospital AKI alert system of the Brandenburg University Hospital. Serum Nostrin, and serum NGAL and KIM-1 were measured within a maximum of 48 hours from the timepoint of initial diagnosis of AKI. The following endpoints were defined: in-hospital death, need of kidney replacement therapy (KRT), recovery of kidney function (ROKF) until discharge. RESULTS: AKI patients had significantly higher serum Nostrin levels compared to Controls. The level of serum Nostrin increased significantly with the severity of AKI. Within the group of AKI patients (n = 150) the in-hospital mortality was 16.7%, KRT was performed in 39.3%, no ROKF occurred in 28%. Patients who required KRT had significantly higher levels of serum Nostrin compared to patients who did not require KRT. Significantly higher levels of serum Nostrin were also detected in AKI patients without ROKF compared to patients with ROKF. In addition, low serum Nostrin levels at the timepoint of AKI diagnosis were predictive of in-hospital survival. For comparison, the serum concentrations of NGAL and KIM-1 were determined in parallel to the Nostrin concentrations and the results confirm the prognostic properties of serum Nostrin in AKI. CONCLUSIONS: The current study suggests serum Nostrin as novel biomarker of AKI-associated mortality, KRT and Acute Kidney Disease.

Association between Neuron-Specific Enolase, Memory Function, and Postoperative Delirium after Transfemoral Aortic Valve Replacement.

INTRODUCTION: Although transfemoral aortic valve replacement (TAVR) is a safe treatment for elderly patients with severe aortic valve stenosis, postoperative microembolism has been described. In this secondary endpoint analysis of the POST-TAVR trial, we aimed to investigate whether changes in neuron-specific enolase (NSE)-a biomarker of neuronal damage-are associated with changes in memory function or postoperative delirium (POD). MATERIALS AND METHODS: This was a prospective single-center study enrolling patients undergoing elective TAVR. Serum NSE was measured before and 24 h after TAVR. POD was diagnosed using CAM-ICU testing. Memory function was assessed before TAVR and before hospital discharge using the "Consortium to Establish a Registry for Alzheimer's Disease" (CERAD) word list and the digit span task (DST) implemented in "∆elta-App". RESULTS: Subjects' median age was 82 years (25th to 75th percentile: 77.5-85.0), 42.6% of subjects were women. CERAD scores significantly increased from pre- to post-TAVR, with p < 0.001. POD occurred in 4.4% (6/135) of subjects at median 2 days after TAVR. After TAVR, NSE increased from a median of 1.85 ng/mL (1.30-2.53) to 2.37 ng/mL (1.69-3.07), p < 0.001. The median increase in NSE was 40.4% (13.1-138.0) in patients with POD versus 17.3% (3.3-43.4) in those without POD (p = 0.17). CONCLUSIONS: Memory function improved after TAVR, likely due to learning effects, with no association to change in NSE. Patients with POD appear to have significantly higher postoperative levels of NSE compared to patients without POD after TAVR. This finding suggests that neuronal damage, as indicated by NSE elevation, may not significantly impair assessed memory function after TAVR.

NT-proBNP/urine hepcidin-25 ratio and cardiorenal syndrome type 1 in patients with severe symptomatic aortic stenosis.

Background: This study aimed to determine whether novel and conventional cardiorenal biomarkers in patients before transcatheter aortic valve implantation may be associated with cardiorenal syndrome (CRS) type 1. Methods: Serum NT-proBNP and urine biomarkers (hepcidin-25, NGAL, IL-6) were measured before and 24 h after transcatheter aortic valve implantation. Results: 16/95 patients had CRS type 1. Those patients had longer length of stay in hospital (12.5 [9.0-16.0] vs 9.0 [8-12] days; p = 0.025) and were more frequently readmitted to hospital within 6 months after discharge (46.7 vs 15.6%; odds ratio: 4.7; 95% CI: 1.5-15.5; p = 0.007). The NT-proBNP/urine hepcidin-25 ratio (odds ratio: 2.89; 95% CI: 1.30-6.41; p = 0.009) was an independent modifier of CRS type 1. Conclusion: The NT-proBNP/urine hepcidin-25 ratio appears to be a modifier of risk of CRS type 1.

Biomarker-Based Prediction of Survival and Recovery of Kidney Function in Acute Kidney Injury.

BACKGROUND: Acute kidney injury (AKI) affects increasing numbers of hospitalized patients; the prognosis remains poor. The diagnosis is still based on the 2012 published KDIGO criteria. Numerous new AKI biomarkers have been identified in recent years; they either reflect impaired excretory function or structural damage. The majority of markers are useful for AKI recognition under certain circumstances. Fewer data are available on the role of biomarkers in the prediction of in-hospital survival and renal recovery post-AKI. The current article is intended to provide information about these two aspects. SUMMARY: The following databases were screened: PubMed, Web of Science, Cochrane Library, Scopus. The period lasted from 2000 until 2022. The following terms were applied: "AKI" AND "biomarker" AND "survival" OR "mortality" OR "recovery of kidney function" OR "renal recovery" OR "kidney recovery". The following terms were used for additional literature search: "TIMP-2" AND "IGFBP7" and "RNA biomarker" AND "hematology". Regarding mortality, exclusively those studies were selected that addressed the in-hospital mortality. Nine (9) studies were identified that evaluated biomarker-based prediction of in-hospital mortality and/or of recovery of kidney function (ROKF). A homogenous definition of ROKF is however missing yet. Currently, some biomarkers, measured early during the course of the disease, are associated with increased mortality risk and/or with a higher chance of renal recovery. KEY MESSAGES: The literature provides only a few biomarker-related studies that address the issues of mortality and recovery. The definition of ROKF needs to be homogenized.

[Depth and integration of science modules in medical studies at recognized public and private faculties in Germany: A cross-sectional study]. / Ausmaß und Integration von Wissenschaftsmodulen in das Medizinstudium an den staatlichen Fakultäten und den privaten staatlich anerkannten Fakultäten in Deutschland: eine Querschnittsstudie.

BACKGROUND: At the end of November 2020, four years after the adoption of the Masterplan Medizinstudium 2020, the Federal Ministry of Health presented the draft bill for the revision of the medical licensing regulations. This was adapted in August 2021 and stipulates, among other things, the completion of a mandatory scientific project up to the second stage of the medical examination, which is to be conducted within twelve weeks and based on the structured teaching of scientific skills using longitudinal courses. This study examines to what extent the mentioned aspects of the draft bill are already included in the current compulsory curricula in German medical study programs. METHODS: In a cross-sectional study, we conducted a document analysis and an online survey of the student deaneries of state and private, state-recognized faculties of medicine in Germany. The objective was to assess the integration of curricular science projects and longitudinal science modules. The data was analyzed descriptively. RESULTS: In total, 40 (93%) of 43 faculties were included in the document analysis. 26 (60.5%) of 43 academic deans responded to the online questionnaire. Only twelve (30%) of the faculties offer a mandatory science project according to the document analysis and eight (30.8%) according to the evaluation of the online survey. In relative terms, a mandatory science project is already a curricular component in the majority of model and reform degree programs (document analysis: 83.3%, n=10 / N=12; questionnaire: 87.5%, n=7 / N=8). This contrasts with a significantly smaller number of regular degree programs that currently offer a science project as a mandatory component (document analysis: 16.7%, n=2 / N=12; questionnaire: 12.5%, n=1 / N=8). In the majority of the model and reform courses, a compulsory (longitudinal) science module has already been integrated into the curriculum (document analysis: 75%, n=12 / N=16; questionnaire: 55.5%, n=6 / N=11). This is only true for 25% (n=4 / N=12; document analysis) and 44.5% (n=5 / N=11; questionnaire) of the regular courses. DISCUSSION: The results of the surveys indicate that the regular study programs, in particular, need to be restructured in order to integrate the intended time frame of twelve weeks for an obligatory science project into the current curriculum. How this is to be achieved without reducing the existing study content seems to be still unclear. Regardless of the current draft bill, however, this step appears to be necessary in order to strengthen the acquisition of scientific competencies in medical studies and to adapt it to international medical education. CONCLUSION: The majority of the current model and reformed medical study courses already fulfill the requirements of the draft bill for the new licensing regulations with regard to the curricular integration of an obligatory scientific project and longitudinal strand on scientific work, which offer hints on design possibilities for further faculties.

The Brandenburg reformed medical curriculum: study locally, work locally.

The Brandenburg Medical School "Theodor Fontane" (MHB) was founded in 2014 by municipal and non-profit institutions in Bernau, Brandenburg an der Havel and Neuruppin to train more physicians for the non-metropolitan region of Brandenburg. Since the 2015 summer term, 48 medical students have been enrolled each year, accepted through the university's own selection process in which the score on the German school-leaving exam (Abitur) and time spent on the waiting list play subordinate roles. Tuition fees can be partially financed through scholarship agreements with regional hospitals if the applicants commit themselves to medical specialist training (Facharztweiterbildung) at a particular hospital. The main places of study are Neuruppin and Brandenburg an der Havel; there is a decentralized study phase from the eighth to tenth semester of study. The Brandenburg Reformed Medical Curriculum (BMM) complies with the model clause contained in the German regulations governing the licensing of medical doctors (ÄAppO). The curriculum is based on problem-based learning (PBL) and focused on competencies and consists of integrated interdisciplinary modules that combine, from the very beginning, basic sciences with clinical and theoretical medical subjects. The focus on general practice is visible in the regularly held "Practical Days" (Praxistag) during which second-year students and above have the opportunity to observe at participating medical practices and familiarize themselves with primary care in Brandenburg. A special focus of BMM is on the acquisition and development of communication and interpersonal skills. These are imparted through a longitudinal curriculum referred to as "Teamwork, Reflection, Interaction, Communication" (TRIK). High value is placed on critical thinking and scientific scholarship and this is reflected in an eight-week academic placement in which the students independently write a research paper. Several different teaching formats ensure that, along with learning specific subjects, sustained personal development can also take place. BMM's decentralized study phase starting in the eighth semester represents a special part of the curriculum in which students complete their clinical training in small groups at selected cooperating hospitals in Brandenburg. This phase encompasses not only hospital placements and other local patient-centered courses, but also centralized instruction via video conferencing to assure that basic sciences and clinical theory continue to be covered. Knowledge- and performance-based semester assessments, in particular OSCEs, reinforce the practical aspects of the training. These replace the M1 state medical examination in the first study phase. The first medical students are now in their ninth semester as of April 2019, making it still too early for final evaluations. The curriculum, successfully implemented to date, already satisfies core requirements of the Master Plan 2020 for undergraduate medical education (Masterplan Medizinstudium 2020) with the curriculum's organization and structure, curricular content, assessment formats and student admission process. With its decentralized structure, BMM specifically addresses the social and health policy challenges facing rural regions of Brandenburg. This is the first curriculum that has taken on the improvement of healthcare in rural regions as its central aim.

Nosip functions during vertebrate eye and cranial cartilage development.

BACKGROUND: The nitric oxide synthase interacting protein (Nosip) has been associated with diverse human diseases including psychological disorders. In line, early neurogenesis of mouse and Xenopus is impaired upon Nosip deficiency. Nosip knockout mice show craniofacial defects and the down-regulation of Nosip in the mouse and Xenopus leads to microcephaly. Until now, the exact underlying molecular mechanisms of these malformations were still unknown. RESULTS: Here, we show that nosip is expressed in the developing ocular system as well as the anterior neural crest cells of Xenopus laevis. Furthermore, Nosip inhibition causes severe defects in eye formation in the mouse and Xenopus. Retinal lamination as well as dorso-ventral patterning of the retina were affected in Nosip-depleted Xenopus embryos. Marker gene analysis using rax, pax6 and otx2 reveals an interference with the eye field induction and differentiation. A closer look on Nosip-deficient Xenopus embryos furthermore reveals disrupted cranial cartilage structures and an inhibition of anterior neural crest cell induction and migration shown by twist, snai2, and egr2. Moreover, foxc1 as downstream factor of retinoic acid signalling is affected upon Nosip deficiency. CONCLUSIONS: Nosip is a crucial factor for the development of anterior neural tissue such the eyes and neural crest cells. Developmental Dynamics 247:1070-1082, 2018. © 2018 Wiley Periodicals, Inc.

Developmental neurogenesis in mouse and Xenopus is impaired in the absence of Nosip.

BACKGROUND: Genetic deletion of Nosip in mice causes holoprosencephaly, however, the function of Nosip in neurogenesis is currently unknown. RESULTS: We combined two vertebrate model organisms, the mouse and the South African clawed frog, Xenopus laevis, to study the function of Nosip in neurogenesis. We found, that size and cortical thickness of the developing brain of Nosip knockout mice were reduced. Accordingly, the formation of postmitotic neurons was greatly diminished, concomitant with a reduced number of apical and basal neural progenitor cells in vivo. Neurospheres derived from Nosip knockout embryos exhibited reduced growth and the differentiation capability into neurons in vitro was almost completely abolished. Mass spectrometry analysis of the neurospheres proteome revealed a reduced expression of Rbp1, a regulator of retinoic acid synthesis, when Nosip was absent. We identified the homologous nosip gene to be expressed in differentiated neurons in the developing brain of Xenopus embryos. Knockdown of Nosip in Xenopus resulted in a reduction of brain size that could be rescued by reintroducing human NOSIP mRNA. Furthermore, the expression of pro-neurogenic transcription factors was reduced and the differentiation of neuronal cells was impaired upon Nosip knockdown. In Xenopus as well as in mouse we identified reduced proliferation and increased apoptosis as underlying cause of microcephaly upon Nosip depletion. In Xenopus Nosip and Rbp1 are similarly expressed and knockdown of Nosip resulted in down regulation of Rbp1. Knockdown of Rbp1 caused a similar microcephaly phenotype as the depletion of Nosip and synergy experiments indicated that both proteins act in the same signalling pathway. CONCLUSIONS: Nosip is a novel factor critical for neural stem cell/progenitor self-renewal and neurogenesis during mouse and Xenopus development and functions upstream of Rbp1 during early neurogenesis.

The F-BAR Protein NOSTRIN Dictates the Localization of the Muscarinic M3 Receptor and Regulates Cardiovascular Function.

RATIONALE: Endothelial dysfunction is an early event in cardiovascular disease and characterized by reduced production of nitric oxide (NO). The F-BAR protein NO synthase traffic inducer (NOSTRIN) is an interaction partner of endothelial NO synthase and modulates its subcellular localization, but the role of NOSTRIN in pathophysiology in vivo is unclear. OBJECTIVE: We analyzed the consequences of deleting the NOSTRIN gene in endothelial cells on NO production and cardiovascular function in vivo using NOSTRIN knockout mice. METHODS AND RESULTS: The levels of NO and cGMP were significantly reduced in mice with endothelial cell-specific deletion of the NOSTRIN gene resulting in diastolic heart dysfunction. In addition, systemic blood pressure was increased, and myograph measurements indicated an impaired acetylcholine-induced relaxation of isolated aortic rings and resistance arteries. We found that the muscarinic acetylcholine receptor subtype M3 (M3R) interacted directly with NOSTRIN, and the latter was necessary for correct localization of the M3R at the plasma membrane in murine aorta. In the absence of NOSTRIN, the acetylcholine-induced increase in intracellular Ca(2+) in primary endothelial cells was abolished. Moreover, the activating phosphorylation and Golgi translocation of endothelial NO synthase in response to the M3R agonist carbachol were diminished. CONCLUSIONS: NOSTRIN is crucial for the localization and function of the M3R and NO production. The loss of NOSTRIN in mice leads to endothelial dysfunction, increased blood pressure, and diastolic heart failure.

The ubiquitin E3 ligase NOSIP modulates protein phosphatase 2A activity in craniofacial development.

Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans.

The F-BAR protein NOSTRIN participates in FGF signal transduction and vascular development.

F-BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F-BAR proteins in vivo. Here we report, that the F-BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.

Platelet-endothelial cell adhesion molecule-1 regulates endothelial NO synthase activity and localization through signal transducers and activators of transcription 3-dependent NOSTRIN expression.

BACKGROUND: NO produced by the endothelial NO synthase (eNOS) is an important regulator of cardiovascular physiological and pathological features. eNOS is activated by numerous stimuli, and its activity is tightly regulated. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in regulating eNOS activity in response to shear stress. The current study was conducted to determine the role of PECAM-1 in the regulation of basal eNOS activity. METHODS AND RESULTS: We demonstrate that PECAM-1-knockout ECs have increased basal eNOS activity and NO production. Mechanistically, increased eNOS activity is associated with a decrease in the inhibitory interaction of eNOS with caveolin-1, impaired subcellular localization of eNOS, and decreased eNOS traffic inducer (NOSTRIN) expression in the absence of PECAM-1. Furthermore, we demonstrate that activation of blunted signal transducers and activators of transcription 3 (STAT3) in the absence of PECAM-1 results in decreased NOSTRIN expression via direct binding of the signal transducers and activators of transcription 3 to the NOSTRIN promoter. CONCLUSIONS: Our results reveal an elegant mechanism of eNOS regulation by PECAM-1 through signal transducers and activators of transcription 3-mediated transcriptional control of NOSTRIN.

Expression and purification of recombinant human EGFL7 protein.

The secreted epidermal growth factor-like protein 7 (EGFL7) plays an important role in angiogenesis, especially in the recruitment of endothelial and smooth muscle cells to the site of the nascent vessel and their ordered assembly into functional vasculature. However, progress in the understanding of the underlying mechanisms is to date greatly hindered by the lack of recombinant EGFL7 protein in a stable, soluble, native state, thus preventing e.g. the characterization of the proposed functional receptor as well as investigation of additional biological effects of EGFL7. So far all attempts to produce sufficient amounts of recombinant EGFL7 protein by various groups have failed. In this study we describe a procedure for the expression and purification of human EGFL7 from Sf9 cells and for the first time provide means to isolate biologically functional EGFL7 protein in sufficient quantities for its further biological characterization. We believe that the availability of EGFL7 will greatly accelerate our understanding of the precise role of EGFL7 and the underlying molecular mechanisms of EGFL7 action in the fundamentally important process of angiogenesis.

Activation of mammalian target of rapamycin controls the loss of TCRzeta in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation.

Persistent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation and death pathway selection in systemic lupus erythematosus. Treatment with rapamycin, which effectively controls disease activity, normalizes CD3/CD28-induced calcium fluxing but fails to influence MHP, suggesting that altered calcium fluxing is downstream or independent of mitochondrial dysfunction. In this article, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in lupus T cells. Activation of mTOR was inducible by NO, a key trigger of MHP, which in turn enhanced the expression of HRES-1/Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes. Expression of HRES-1/Rab4 was increased in CD4(+) lupus T cells, and in accordance with its dominant impact on the endocytic recycling of CD4, it was inversely correlated with diminished CD4 expression. HRES-1/Rab4 overexpression was also inversely correlated with diminished TCRzeta protein levels. Pull-down studies revealed a direct interaction of HRES-1/Rab4 with CD4 and TCRzeta. Importantly, the deficiency of the TCRzeta chain and of Lck and the compensatory up-regulation of FcepsilonRIgamma and Syk, which mediate enhanced calcium fluxing in lupus T cells, were reversed in patients treated with rapamcyin in vivo. Knockdown of HRES-1/Rab4 by small interfering RNA and inhibitors of lysosomal function augmented TCRzeta protein levels in vitro. The results suggest that activation of mTOR causes the loss of TCRzeta in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.

NOSTRINbeta--a shortened NOSTRIN variant with a role in transcriptional regulation.

We recently observed that a novel, shortened variant of eNOS trafficking inducer (NOSTRIN) is expressed in cirrhotic liver. This shortened variant (NOSTRINbeta) lacks the first 78 amino acids of full-length NOSTRIN (NOSTRINalpha) and thus a substantial part of its F-BAR domain. In contrast to NOSTRINalpha, NOSTRINbeta mainly localizes to the cell nucleus. In this study, we show that nuclear import of NOSTRINbeta depends on two nuclear localization signals (aa 32-36: KKRK and aa 57-61: KAKKK). Each of the sequences is independently functional, but both are required to sustain nuclear localization of NOSTRINbeta. Export of NOSTRINbeta from the nucleus is facilitated by a CRM1-dependent mechanism relying on the nuclear export sequence LELEKERIQL (aa 135-145). Unlike NOSTRINbeta, the full-length variant NOSTRINalpha was conspicuously absent from the nucleus. This is most likely because of the fact that its N-terminal F-BAR domain, which is truncated in NOSTRINbeta, facilitates association with cellular membranes. NOSTRINbeta directly binds to the 5'-regulatory region of the NOSTRIN gene (bp -200 to -1), and overexpression of NOSTRINbeta strongly decreases transcription of a reporter gene under control of this DNA region. Taken together, our results suggest that nuclear NOSTRINbeta may negatively regulate transcription of the NOSTRIN gene.

The iromycins, a new family of pyridone metabolites from Streptomyces sp. I. Structure, NOS inhibitory activity, and biosynthesis.

The iromycins A-D are members of a new family of rare alpha-pyridone metabolites. The isolation and structure elucidation of these microbial secondary metabolites from Streptomyces sp. Dra 17 revealed a N-heterocyclic core structure with two unusual side chains. Iromycins act as inhibitors of nitric oxide synthases (NOS), a protein family, which produces the crucial second messenger nitric oxide (NO). Importantly, these compounds inhibit selectively endothelial NOS rather than neuronal NOS and thus set prospects for both medical therapy and basic research. Feeding experiments with 13C- and 15N -labeled precursors indicated an uncommon type of polyketide biosynthesis and clearly ruled out an isoprenoid origin. A detoxification pathway of a particular secondary metabolite in the host strain is a rare observation and here we demonstrate it with the iromycin family.

Translocation of endothelial nitric-oxide synthase involves a ternary complex with caveolin-1 and NOSTRIN.

Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOS-trafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1-61) and the central domain of NOSTRIN (positions 323-434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell.

Subcellular targeting and trafficking of nitric oxide synthases.

Unlike most other endogenous messengers that are deposited in vesicles, processed on demand and/or secreted in a regulated fashion, NO (nitric oxide) is a highly active molecule that readily diffuses through cell membranes and thus cannot be stored inside the producing cell. Rather, its signalling capacity must be controlled at the levels of biosynthesis and local availability. The importance of temporal and spatial control of NO production is highlighted by the finding that differential localization of NO synthases in cardiomyocytes translates into distinct effects of NO in the heart. Thus NO synthases belong to the most tightly controlled enzymes, being regulated at transcriptional and translational levels, through co- and post-translational modifications, by substrate availability and not least via specific sorting to subcellular compartments, where they are in close proximity to their target proteins. Considerable efforts have been made to elucidate the molecular mechanisms that underlie the intracellular targeting and trafficking of NO synthases, to ultimately understand the cellular pathways controlling the formation and function of this powerful signalling molecule. In the present review, we discuss the mechanisms and triggers for subcellular routing and dynamic redistribution of NO synthases and the ensuing consequences for NO production and action.