Increased Intestinal Permeability and Decreased Resiliency of the Intestinal Barrier in Alcoholic Liver Disease.

INTRODUCTION: Only 20%-30% of individuals with alcohol use disorder (AUD) develop alcoholic liver disease (ALD). While the development of gut-derived endotoxemia is understood to be a required cofactor, increased intestinal permeability in ALD is not completely understood. METHODS: We recruited 178 subjects-58 healthy controls (HCs), 32 with ALD, 53 with AUD but no liver disease (ALC), and 35 with metabolic dysfunction-associated steatotic liver disease (MASLD). Intestinal permeability was assessed by a sugar cocktail as a percentage of oral dose. The permeability test was repeated after an aspirin challenge in a subset. RESULTS: Five-hour urinary lactulose/mannitol ratio (primarily representing small intestinal permeability) was not statistically different in HC, ALC, ALD, and MASLD groups ( P = 0.40). Twenty-four-hour urinary sucralose (representing whole gut permeability) was increased in ALD ( F = 5.3, P < 0.01) and distinguished ALD from ALC; 24-hour sucralose/lactulose ratio (primarily representing colon permeability) separated the ALD group ( F = 10.2, P < 0.01) from the MASLD group. After aspirin challenge, intestinal permeability increased in all groups and ALD had the largest increase. DISCUSSION: In a group of patients, we confirmed that (i) the ALD group has increased intestinal permeability compared with the HC, ALC, or MASLD group. In addition, because small bowel permeability (lactulose/mannitol ratio) is normal, the disruption of intestinal barrier seems to be primarily in the large intestine; (ii) decreased resiliency of intestinal barrier to injurious agents (such as NSAID) might be the mechanism for gut leak in subset of AUD who develop ALD.

Oral mannitol for bowel preparation: a dose-finding phase II study.

BACKGROUND: Successful bowel preparation (BP) for colonoscopy depends on the instructions, diet, the laxative product, and patient adherence, which all affect colonoscopy quality. Nevertheless, there are no laxatives which combine effectiveness, safety, easy self-administration, good patient acceptance, and low cost. However, mannitol, a sugar alcohol, could be an attractive candidate for use in clinical practice if it is shown to demonstrate adequate efficacy and safety. AIMS: The present phase II dose-finding study compared three doses of mannitol (50, 100, and 150 g) to identify the best dose to be used in a subsequent phase III study. METHODS: The Boston Bowel Preparation Scale, caecal intubation rate, adherence, acceptability, and safety profile, including measurement of potentially dangerous colonic gas concentrations (CH4, H2, O2), were considered in all patients. A weighted algorithm was used to identify the best mannitol dose for use in the subsequent study. RESULTS: The per-protocol population included 60 patients in the 50 g group, 54 in the 100 g group, and 49 in the 150 g group. The 100 g dose was the best as it afforded optimal colon cleansing efficacy (94.4% of patients had adequate BP), adherence, acceptability, and safety, including negligible gas concentrations. CONCLUSIONS: The present study demonstrated that the colon cleansing efficacy and safety of mannitol were dose dependent. Conversely, gas concentrations were not dose dependent and negligible in all patients. Combined evaluation of efficacy, tolerability, and safety, using a weighted algorithm, determined that mannitol 100 g was the best dose for the phase III study.

A Semi-Physiological Three-Compartment Model Describes Brain Uptake Clearance and Efflux of Sucrose and Mannitol after IV Injection in Awake Mice.

PURPOSE: To evaluate a three-compartmental semi-physiological model for analysis of uptake clearance and efflux from brain tissue of the hydrophilic markers sucrose and mannitol, compared to non-compartmental techniques presuming unidirectional uptake. METHODS: Stable isotope-labeled [13C]sucrose and [13C]mannitol (10 mg/kg each) were injected as IV bolus into the tail vein of awake young adult mice. Blood and brain samples were taken after different time intervals up to 8 h. Plasma and brain concentrations were quantified by UPLC-MS/MS. Brain uptake clearance (Kin) was analyzed using either the single-time point analysis, the multiple time point graphical method, or by fitting the parameters of a three-compartmental model that allows for symmetrical exchange across the blood-brain barrier and an additional brain efflux clearance. RESULTS: The three-compartment model was able to describe the experimental data well, yielding estimates for Kin of sucrose and mannitol of 0.068 ± 0.005 and 0.146 ± 0.020 µl.min-1.g-1, respectively, which were significantly different (p < 0.01). The separate brain efflux clearance had values of 0.693 ± 0.106 (sucrose) and 0.881 ± 0.20 (mannitol) µl.min-1.g-1, which were not statistically different. Kin values obtained by single time point and multiple time point analyses were dependent on the terminal sampling time and showed declining values for later time points. CONCLUSIONS: Using the three-compartment model allows determination of Kin for small molecule hydrophilic markers with low blood-brain barrier permeability. It also provides, for the first time, an estimate of brain efflux after systemic administration of a marker, which likely represents bulk flow clearance from brain tissue.

Hyperosmolar blood-brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance.

The blood-brain barrier (BBB) is the main obstacle to the effective delivery of therapeutic agents to the brain, compromising treatment efficacy for a variety of neurological disorders. Intra-arterial (IA) injection of hyperosmotic mannitol has been used to permeabilize the BBB and improve parenchymal entry of therapeutic agents following IA delivery in preclinical and clinical studies. However, the reproducibility of IA BBB manipulation is low and therapeutic outcomes are variable. We demonstrated that this variability could be highly reduced or eliminated when the procedure of osmotic BBB opening is performed under the guidance of interventional MRI. Studies have reported the utility and applicability of this technique in several species. Here we describe a protocol to open the BBB by IA injection of hyperosmotic mannitol under the guidance of MRI in mice. The procedures (from preoperative preparation to postoperative care) can be completed within ~1.5 h, and the skill level required is on par with the induction of middle cerebral artery occlusion in small animals. This MRI-guided BBB opening technique in mice can be utilized to study the biology of the BBB and improve the delivery of various therapeutic agents to the brain.

Del Nido versus HTK cardioplegia for myocardial protection during adult complex valve surgery: a retrospective study.

BACKGROUND: Histidine-tryptophan-ketoglutarate (HTK) and del Nido (DN) cardioplegia are intracellular-type and extracellular-type solution respectively, both can provide a long period of myocardial protection with single-dose infusion, but studies comparing the two are rare for adult cardiac surgery. This study aims to evaluate whether DN is suitable for cardioplegia in complex and high-risk valve surgery with long-term cardiac ischemia when compared with HTK. METHODS: The perioperative records of adult patients infused with DN/HTK as a cardioplegic solution who underwent complex valve surgery with an expected myocardial ischaemic duration longer than 90 min between Oct 2018 and Oct 2019 were analysed retrospectively. RESULTS: Of the 160 patients who received DN/HTK and underwent complex valve surgery, we propensity matched 73 pairs. Both groups achieved satisfactory cardiac arrest effects, and no significant difference was found in their cTnI and CK-MB levels within 12 to 72 h postoperatively. The DN group had a higher rate of return to spontaneous rhythm (0.88 v 0.52, P < 0.001), a lower frequency of postoperative severe arrythmias (12% v 26%, P = 0.036), a higher postoperative stroke volume (65 v 59 ml, P = 0.011) and a higher cardiac output (6.0 v 4.9 L/min, P = 0.007) as evaluated by echocardiography, fewer transfusions and shorter ICU stays (both P < 0.05). The two groups had similar inotrope usage and similar incidences of low cardiac output, morbidities and mortality. Subgroup analysis showed that when the aortic clamping time was greater than 120 min, the advantages of DN were weakened. CONCLUSIONS: DN can be safely applied to complex valve surgery, and it has a similar myocardial protection effect as HTK. Further prospective studies are required to verify these retrospective findings. Trial registration retrospectively registered.

The use of the mannitol test as an outcome measure in asthma intervention studies: a review and practical recommendations.

BACKGROUND: The mannitol test is an indirect bronchial challenge test widely used in diagnosing asthma. Response to the mannitol test correlates with the level of eosinophilic and mast cell airway inflammation, and a positive mannitol test is highly predictive of a response to anti-inflammatory treatment with inhaled corticosteroids. The response to mannitol is a physiological biomarker that may, therefore, be used to assess the response to other anti-inflammatory treatments and may be of particular interest in early phase studies that require surrogate markers to predict a clinical response. The main objectives of this review were to assess the practical aspects of using mannitol as an endpoint in clinical trials and provide the clinical researcher and respiratory physician with recommendations when designing early clinical trials. METHODS: The aim of this review was to summarise previous uses of the mannitol test as an outcome measure in clinical intervention studies. The PubMed database was searched using a combination of MeSH and keywords. Eligible studies included intervention or repeatability studies using the standard mannitol test, at multiple timepoints, reporting the use of PD15 as a measure, and published in English. RESULTS: Of the 193 papers identified, 12 studies met the inclusion criteria and data from these are discussed in detail. Data on the mode of action, correlation with airway inflammation, its diagnostic properties, and repeatability have been summarised, and suggestions for the reporting of test results provided. Worked examples of power calculations for dimensioning study populations are presented for different types of study designs. Finally, interpretation and reporting of the change in the response to the mannitol test are discussed. CONCLUSIONS: The mechanistic and practical features of the mannitol test make it a useful marker of disease, not only in clinical diagnoses, but also as an outcome measure in intervention trials. Measuring airway hyperresponsiveness to mannitol provides a novel and reproducible test for assessing efficacy in intervention trials, and importantly, utilises a test that links directly to underlying drivers of disease.

Absorption, metabolism and excretion of once-weekly somapacitan, a long-acting growth hormone derivative, after single subcutaneous dosing in human subjects.

Somapacitan is a reversible albumin-binding growth hormone (GH) derivative in clinical development for once-weekly administration in patients with adult GH deficiency (AGHD) and children with GH deficiency (GHD). To date, the use of somapacitan in AGHD or severe AGHD has been approved in the USA and Japan, respectively. This study (ClinicalTrials.gov, NCT02962440) investigated the absorption, metabolism and excretion, as well as the pharmacokinetics (PK), of tritium-labelled somapacitan ([3H]-somapacitan). Seven healthy males received a single subcutaneous dose of 6 mg somapacitan containing [3H]-somapacitan 20 MBq. Blood, serum, plasma, urine, faeces, and expired air were collected for radioactivity assessment. Metabolites were identified and quantified in plasma and urine collected. The PK of plasma components were determined, and the radioactive peaks of the most abundant plasma metabolites and urine metabolites were selected for analysis. Twenty-eight days after dosing, 94.0% of the administered dose was recovered as [3H]-somapacitan-related material, most of which was excreted in urine (80.9%); 12.9% was excreted in faeces, and an insignificant amount (0.2%) was exhaled in expired air. PK properties of [3H]-somapacitan-related material appeared to be consistent across plasma, serum and blood. Three abundant plasma metabolites (P1, M1 and M1B) and two abundant urine metabolites (M4 and M5) were identified. The total exposure of intact somapacitan accounted for 59% of the total exposure of all somapacitan-related material, P1 accounted for 21% and M1 plus M1B accounted for 12%. M4 and M5 were the most abundant urine metabolites and accounted for 37% and 8% of the dosed [3H]-somapacitan radioactivity, respectively. No intact somapacitan was found in excreta. Two subjects had six adverse events (AEs); all were mild in severity and unlikely to be related to trial product. The majority of dosed [3H]-somapacitan (94%) was recovered as excreted metabolites. Urine was the major route for excretion of somapacitan metabolites, followed by faeces, and exhalation in expired air was negligible. The low molecular weights of identified urine metabolites demonstrate that somapacitan was extensively degraded to small residual fragments that were excreted (fully biodegradable). The extensive metabolic degradation and full elimination of metabolites in excreta were the major clearance pathways of somapacitan and the key elements in its biological fate. A single dose of 6 mg somapacitan (containing [3H]-somapacitan) in healthy male subjects was well tolerated with no unexpected safety issues identified.

Spray-Dried Inhalable Powder Formulations of Therapeutic Proteins and Peptides.

Respiratory diseases are among the leading causes of morbidity and mortality worldwide. Innovations in biochemical engineering and understanding of the pathophysiology of respiratory diseases resulted in the development of many therapeutic proteins and peptide drugs with high specificity and potency. Currently, protein and peptide drugs are mostly administered by injections due to their large molecular size, poor oral absorption, and labile physicochemical properties. However, parenteral administration has several limitations such as frequent dosing due to the short half-life of protein and peptide in blood, pain on administration, sterility requirement, and poor patient compliance. Among various noninvasive routes of administrations, the pulmonary route has received a great deal of attention and is a better alternative to deliver protein and peptide drugs for treating respiratory diseases and systemic diseases. Among the various aerosol dosage forms, dry powder inhaler (DPI) systems appear to be promising for inhalation delivery of proteins and peptides due to their improved stability in solid state. This review focuses on the development of DPI formulations of protein and peptide drugs using advanced spray drying. An overview of the challenges in maintaining protein stability during the drying process and stabilizing excipients used in spray drying of proteins and peptide drugs is discussed. Finally, a summary of spray-dried DPI formulations of protein and peptide drugs, their characterization, various DPI devices used to deliver protein and peptide drugs, and current clinical status are discussed.

Immune response and protective efficacy of two new adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02, administered with a Streptococcus agalactiae ghost vaccine in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCRß, IL-1ß, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

Delayed transient obstructive hydrocephalus after cerebral aneurysm rupture: A case report.

RATIONALE: Obstructive hydrocephalus (OH) frequently occurs in patients with a ruptured cerebral aneurysm (CA), and it may lead to severe neurological deficits, including life-threatening brain herniation. OH generally occurs in the early stage of CA rupture, rather than in the late stage, and rarely resolves without therapy. PATIENT CONCERNS: A 64-year-old woman with a ruptured anterior communicating artery aneurysm was treated with coil embolization. Nineteen days after her CA rupture, because of the delayed transient OH, she experienced a dramatic cycle in consciousness over 9 hours: wakefulness-drowsiness-coma-drowsiness-wakefulness. DIAGNOSIS: The patient was diagnosed with delayed transient obstructive hydrocephalus, which is a very rare condition. INTERVENTIONS: Mannitol was administered to reduce intracranial pressure. OUTCOMES: The patient was discharged from the hospital 30 days after admission, with a final GCS score of 15 and without weaknesses. At follow-up 2 months after discharge, brain CT revealed non-recurrence of hydrocephalus. LESSONS: A blood clot of any size in the ventricle is likely to lead to obstructive hydrocephalus. Prolonged bed rest for IVH patients may help to reduce the incidence of delayed OH.

Spray-Congealing and Wet-Sieving as Alternative Processes for Engineering of Inhalation Carrier Particles: Comparison of Surface Properties, Blending and In Vitro Performance.

PURPOSE: Traditionally, α-lactose monohydrate is the carrier of choice in dry powder inhaler (DPI) formulations. Nonetheless, other sugars, such as D-mannitol, have emerged as potential alternatives. Herein, we explored different particle engineering processes to produce D-mannitol carriers for inhaled delivery. METHODS: Wet-sieving and spray-congealing were employed as innovative techniques to evaluate the impact of engineering on the particle properties of D-mannitol. To that end, the resulting powders were characterized concerning their solid-state, micromeritics and flowability. Afterwards, the engineered carrier particles were blended with inhalable size beclomethasone dipropionate to form low dose (1 wt%) DPI formulations. The in vitro aerosolization performance was evaluated using the NEXThaler®, a reservoir multi-dose device. RESULTS: Wet-sieving generated D-mannitol particles with a narrow particle size distribution and spray-congealing free-flowing spherical particles. The more uniform pumice particles with deep voids and clefts of wet-sieved D-mannitol (Pearl300_WS) were beneficial to drug aerosolization, only when used in combination with a ternary agent (10 wt% of 'Preblend'). When compared to the starting material, the spray-congealed D-mannitol has shown to be promising in terms of the relative increase of the fine particle fraction of the drug (around 100%), when used without the addition of ternary agents. CONCLUSIONS: The wet-sieving process and the related aerosolization performance are strongly dependent on the topography and structure of the starting material. Spray-congealing, has shown to be a potential process for generating smooth spherical particles of D-mannitol that enhance the in vitro aerosolization performance in binary blends of the carrier with a low drug dose.

Delayed pneumocephalus induced by repeated percutaneous aspiration after spinal surgery: A case report and literature review.

RATIONALE: Severe tension pneumocephalus can lead to drowsiness, coma, and even brain hernia and death. The occurrence of delayed pneumocephalus after spinal surgery is rarely reported and often ignored. Herein, we report a case of delayed pneumocephalus after repeated percutaneous aspiration following spinal surgery. PATIENT CONCERNS: A 55-year-old man was admitted in October 2020 because of aggravation in bilateral lower limb weakness and dysuria for seven days. He was diagnosed with liver cancer a year ago, and he underwent several operations because of tumor recurrence. The patient underwent thoracic vertebrae tumor excision on this admission, and no cerebrospinal fluid leakage was discovered during surgery. After the third drainage by percutaneous aspiration, the patient complained of severe headache and vomiting on postoperative day 16. DIAGNOSIS: Emergency brain computed tomography revealed massive pneumocephalus. INTERVENTIONS: Thereafter, suction drainage was discontinued, and he was placed on bed rest and administered intravenous mannitol. OUTCOMES: Repeated computed tomography showed complete resolution of the pneumocephalus after five days. LESSONS: Wound exudates and cystic fluid after spinal surgery should be differentiated from cerebrospinal fluid leakage. Reckless percutaneous aspirations can form pneumocephalus in patients with an occult dural injury, and pneumocephalus can occur up to 16 days after surgery. Early diagnosis of pneumocephalus is crucial to avoid severe consequences.

Randomized, Double-Blind, Placebo-Controlled Study of Poly-L-Lactic acid for Treatment of Cellulite in the Lower Extremities.

BACKGROUND: Poly-l-lactic acid (PLLA) is an injectable volumizer with biostimulatory properties used for volumetric structural rejuvenation in patients with facial fat volume loss but has increasingly been utilized for off-face applications. OBJECTIVE: The objectives of this randomized, double-blind, placebo-controlled single center study was to assess the safety and effectiveness of PLLA for the treatment of lower extremity cellulite in adult women. METHODS: 31 healthy women were enrolled in the study. Eligible subjects received 3 treatments every 4 weeks with either PLLA (treatment group) or saline (control group) injections combined with subcision, into each of the glutes or thighs. Follow-up visits were at 1, 3, and 6 months after treatment. Assessments included live ratings, rating of standardized pictures by a blinded evaluator, patient questionnaires, safety, and tolerability ratings. RESULTS: At the 3 and 6-month follow-up, there was a statistically significant change in the global aesthetic improvement scale (GAIS) compared to baseline as assessed by blinded investigators. Significant improvements were shown in the cellulite severity scale (CSS) as well as in the subject satisfaction questionnaires. Treatments were found to be tolerable, and no severe treatment-related adverse events occurred. CONCLUSION: Repeated PLLA treatments combined with subcision are effective and safe in improving the appearance of cellulite. J Drugs Dermatol. 20(5): doi:10.36849/JDD.5380.

Formulation of HBsAg in Montanide ISA 51VG adjuvant: Immunogenicity study and monitoring long-lived humoral immune responses.

Montanide ISA 51VG adjuvant has been approved for human clinical application and stimulates cellular and humoral immune responses. Here, HBsAg was formulated in Montanide ISA51VG adjuvant to compare its potency with the Fendrix and HBsAg-alum vaccines. In particular, the long-term humoral response was assessed up to 220 days after the final immunization. BALB/c mice were allocated into six groups. Treatment groups were injected with HBsAg-Montanide ISA51VG, the Fendrix and commercial HBsAg-alum, respectively. Montanide ISA51 VG, Alum and PBS injected mice were considered as control groups. Mice were immunized three times with 2-week intervals on days 0, 14 and 28 by subcutaneous injection. Lymphocyte proliferation was assessed with the BrdU method. IFN-γ, IL-2 and IL-4 cytokines, specific total IgG and IgG1/IgG2a isotypes were assessed using ELISA. The HBsAg-Montanide ISA51VG vaccine resulted in a significant increase in lymphocyte proliferation versus HBsAg-alum and higher IL-2 cytokine production versus the Fendrix. Comparable IL-4 and IFN-γ cytokines responses were observed for these vaccines. Following the first immunization, IgG increased more in HBs-Montanide 51VG group versus the HBs-alum group, while after the second and third shots comparable responses were observed in comparison to the HBs-alum group. Monitoring for 220 days after the final vaccination showed the superiority of HBsAg-Montanide ISA 51VG vaccine versus HBsAg-alum and even the Fendrix vaccine in the induction of long-term antibody responses. This study suggests that HBsAg-Montanide ISA51VG as a novel vaccine formulation can trigger both cellular and long-lasting humoral immune responses more efficiently than conventional HBsAg vaccines.

Efficacy and safety of inhaled dry-powder mannitol in adults with cystic fibrosis: An international, randomized controlled study.

BACKGROUND: Mannitol is a mucoactive hyperosmotic agent used as add-on therapy in patients with cystic fibrosis (CF), administered twice-daily (BID) via a small, portable, breath-actuated dry-powder inhaler. This study was conducted to provide confirmatory evidence of mannitol's efficacy and safety in adults. METHODS: This multicenter, double-blind, randomized, parallel-group, controlled clinical trial recruited adults (aged ≥18 years) with CF, and forced expiratory volume in 1 second (FEV1) 40-90% predicted. Subjects received either mannitol 400 mg or mannitol 50 mg (control), BID via dry-powder inhaler for 26 weeks. Primary endpoint: FEV1 averaged over the 26-week treatment period. RESULTS: Of 423 subjects randomized (209 or 214 receiving mannitol 400 mg BID or control, respectively), 373 (88.2%) completed the study, with a similar proportion completing in the two groups. For FEV1 averaged over 26 weeks, mannitol 400 mg BID was statistically superior to control (adjusted mean difference 54 mL [95% CI 8, 100 mL]; p = 0.020). This was supported by sensitivity analyses of the primary endpoint, and by observed improvements in secondary pulmonary function endpoints (eg, absolute adjusted mean difference in percent predicted FEV1 averaged over 26 weeks 1.21% [0.07%, 2.36%]; p = 0.037). Adverse events were mainly mild or moderate in severity, with treatment-related adverse events in 15.5 and 12.2% of subjects receiving mannitol 400 mg BID and control, respectively. CONCLUSIONS: In adults with CF, mannitol 400 mg BID inhaled as a dry-powder statistically significantly improved lung function (FEV1) compared with control, with this improvement supported by sensitivity analyses and secondary pulmonary function endpoints. Mannitol had a good overall safety and tolerability profile. ClinicalTrials.gov: NCT02134353.

Hyperosmolarity stimulates transporter-mediated insertion of estrone sulfate into the plasma membrane, but inhibits the uptake by SLC10A1 (NTCP).

Many drugs are largely hydrophobic molecules; a transporter might conceivably insert these into the plasma membrane. At least 18 transporters from diverse families have been reported to transport the model compound estrone sulfate alias estrone-3-sulfate (E3S). Out of these, we recently examined SLC22A11 (OAT4). We concluded from a comparison of E3S and uric acid transport that SLC22A11 does not translocate E3S into the cytosol, but into the plasma membrane. Here we present a hyperosmolarity alias hypertonicity assay to differentiate transport mechanisms. Human transporters were expressed heterologously in 293 cells. Solute uptake into intact cells was measured by LC-MS. Addition of mannitol or sucrose led to rapid cell shrinkage, but cell viability after 60 min in hyperosmolar buffer was not impaired. A decrease in substrate accumulation with increasing osmolarity as observed here for several substrates and the transporters SLC22A11, ETT (SLC22A4), OCT2 (SLC22A2), OAT3 (SLC22A8), and MATE1 (SLC47A1) suggests regular substrate translocation into the cytosol. An increase as observed for E3S transport by SLC22A11, OAT3, MATE1, SLC22A9, and SLC10A6 implies insertion into the membrane. In marked contrast to the other E3S transporters, the bile acid transporter SLC10A1 (NTCP, Na+ taurocholate co-transporting polypeptide) showed a decrease in the accumulation of E3S in hyperosmolar buffer; the same was observed with taurocholic acid. Indeed, our data from several functional assays strongly suggest that the transport mechanism is identical for both substrates. Apparently, a unique transport mechanism has been established for SLC10A1 by evolution that ensures the transport of amphipathic, detergent-like molecules into the cytosol.

Acute glucose influx-induced mitochondrial hyperpolarization inactivates myosin phosphatase as a novel mechanism of vascular smooth muscle contraction.

It is well-established that long-term exposure of the vasculature to metabolic disturbances leads to abnormal vascular tone, while the physiological regulation of vascular tone upon acute metabolic challenge remains unknown. Here, we found that acute glucose challenge induced transient increases in blood pressure and vascular constriction in humans and mice. Ex vivo study in isolated thoracic aortas from mice showed that glucose-induced vascular constriction is dependent on glucose oxidation in vascular smooth muscle cells. Specifically, mitochondrial membrane potential (ΔΨm), an essential component in glucose oxidation, was increased along with glucose influx and positively regulated vascular smooth muscle tone. Mechanistically, mitochondrial hyperpolarization inhibited the activity of myosin light chain phosphatase (MLCP) in a Ca2+-independent manner through activation of Rho-associated kinase, leading to cell contraction. However, ΔΨm regulated smooth muscle tone independently of the small G protein RhoA, a major regulator of Rho-associated kinase signaling. Furthermore, myosin phosphatase target subunit 1 (MYPT1) was found to be a key molecule in mediating MLCP activity regulated by ΔΨm. ΔΨm positively phosphorylated MYPT1, and either knockdown or knockout of MYPT1 abolished the effects of glucose in stimulating smooth muscle contraction. In addition, smooth muscle-specific Mypt1 knockout mice displayed blunted response to glucose challenge in blood pressure and vascular constriction and impaired clearance rate of circulating metabolites. These results suggested that glucose influx stimulates vascular smooth muscle contraction via mitochondrial hyperpolarization-inactivated myosin phosphatase, which represents a novel mechanism underlying vascular constriction and circulating metabolite clearance.