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Background: Long-lasting local anesthetic use for perioperative pain control is limited by possible cardiotoxicity (e.g., arrhythmias and contractile depression), potentially leading to cardiac arrest. Off-target cardiac sodium channel blockade is considered the canonical mechanism behind cardiotoxicity; however, it does not fully explain the observed toxicity variability between anesthetics. The authors hypothesize that more cardiotoxic anesthetics (e.g., bupivacaine) differentially perturb other important cardiomyocyte functions (e.g., calcium dynamics), which may be exploited to mitigate drug toxicity., Methods: The authors investigated the effects of clinically relevant concentrations of racemic bupivacaine, levobupivacaine, or ropivacaine on human stem cell-derived cardiomyocyte tissue function. Contractility, rhythm, electromechanical coupling, field potential profile, and intracellular calcium dynamics were quantified using multielectrode arrays and optical imaging. Calcium flux differences between bupivacaine and ropivacaine were probed with pharmacologic calcium supplementation or blockade. In vitro findings were correlated in vivo using an anesthetic cardiotoxicity rat model (females; n = 5 per group)., Results: Bupivacaine more severely dysregulated calcium dynamics than ropivacaine in vitro (e.g., contraction calcium amplitude to 52 ± 11% and calcium-mediated repolarization duration to 122 ± 7% of ropivacaine effects, model estimate ± standard error). Calcium supplementation improved tissue contractility and restored normal beating rhythm (to 101 ± 6%, and 101 ± 26% of control, respectively) for bupivacaine-treated tissues, but not ropivacaine (e.g., contractility at 80 ± 6% of control). Similarly, calcium pretreatment mitigated anesthetic-induced arrhythmias and cardiac depression in rats, improving animal survival for bupivacaine by 8.3 ± 2.4 min, but exacerbating ropivacaine adverse effects (reduced survival by 13.8 ± 3.4 min and time to first arrhythmia by 12.0 ± 2.9 min). Calcium channel blocker nifedipine coadministration with bupivacaine, but not ropivacaine, exacerbated cardiotoxicity, supporting the role of calcium flux in differentiating toxicity., Conclusions: Our data illustrate differences in calcium dynamics between anesthetics and how calcium may mitigate bupivacaine cardiotoxicity. Moreover, our findings suggest that bupivacaine cardiotoxicity risk may be higher than for ropivacaine in a calcium deficiency context., (Copyright © 2022, the American Society of Anesthesiologists. All Rights Reserved.)

Background: Aryl hydrocarbon receptor (AhR) plays important roles in modulating immune responses. However, the role of AhR in rat liver transplantation (LT) has not been explored., Methods: Safety and side effects of N -(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) and 2-methyl-2H-pyrazole-3-carboxylic acid amide (CH223191) were evaluated. We used optimal doses of 2 drugs, 3,4-DAA, a drug used for mediating AhR activation, and CH223191, antagonist of AhR (3,4-DAA, CH223191, and 3,4-DAA + CH223191), intraperitoneally administered to recipients daily to investigate the role of AhR in the rat LT model. The recipient livers were used to observe the pathological changes, the cells infiltrating the graft, and changes of AhR and programmed death-1 (PD-1) by Western blot, real-time polymerase chain reaction, and immunofluorescence assays. The contents of Foxp3 + and PD-1 + T cells in the recipient spleen and peripheral blood mononuclear cells were evaluated by flow cytometry. In vitro, after isolating CD4 + T cells, they were treated with different AhR ligands to observe the differentiation direction and PD-1 expression level., Results: The activation of AhR by 3,4-DAA prolonged survival time and ameliorated graft rejection, which were associated with increased expression of AhR and PD-1 in the livers and increased Foxp3 + T cells and PD-1 + T cells in recipient spleens, livers, and peripheral blood mononuclear cells. In vitro, primary T cells incubated with 3,4-DAA mediated increased proportion of Treg and PD-1 + T cells. However, the suppression of AhR with CH223191 reverses these effects, both in the LT model and in vitro., Conclusions: Our results indicated that AhR activation might reduce the occurrence of rat acute rejection by increasing the proportion of Treg and the expression of PD-1., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The development of thoracic aortic dissection (TAD) is closely related to extracellular matrix degradation and vascular smooth muscle cell (VSMC) transformation from contractile to synthetic type. LGMN (legumain) degrades extracellular matrix components directly or by activating downstream signals. The role of LGMN in VSMC differentiation and the occurrence of TAD remains elusive., Methods: Microarray datasets concerning vascular dissection or aneurysm were downloaded from the Gene Expression Omnibus database to screen differentially expressed genes. Four-week-old male Lgmn knockout mice (Lgmn -/- ), macrophage-specific Lgmn knockout mice (Lgmn F/F ;LysM Cre ), and RR-11a-treated C57BL/6 mice were given BAPN (β-aminopropionitrile monofumarate; 1 g/kg/d) in drinking water for 4 weeks for TAD modeling. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Cell interaction was examined in macrophage and VSMC coculture system. The reciprocity of macrophage-derived LGMN with integrin αvβ3 in VSMCs was tested by coimmunoprecipitation assay and colocalization analyses., Results: Microarray datasets from the Gene Expression Omnibus database indicated upregulated LGMN in aorta from patients with TAD and mice with angiotensin II-induced AAA. Elevated LGMN was evidenced in aorta and sera from patients with TAD and mice with BAPN-induced TAD. BAPN-induced TAD progression was significantly ameliorated in Lgmn-deficient or inhibited mice. Macrophage-specific deletion of Lgmn alleviated BAPN-induced extracellular matrix degradation. Unbiased profiler polymerase chain reaction array and Gene Ontology analysis displayed that LGMN regulated VSMC phenotype transformation. Macrophage-specific deletion of Lgmn ameliorated VSMC phenotypic switch in BAPN-treated mice. Macrophage-derived LGMN inhibited VSMC differentiation in vitro as assessed by macrophages and the VSMC coculture system. Macrophage-derived LGMN bound to integrin αvβ3 in VSMCs and blocked integrin αvβ3, thereby attenuating Rho GTPase activation, downregulating VSMC differentiation markers and eventually exacerbating TAD development. ROCK (Rho kinase) inhibitor Y-27632 reversed the protective role of LGMN depletion in vascular dissection., Conclusions: LGMN signaling may be a novel target for the prevention and treatment of TAD.

Purpose: To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits., Methods: UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds., Results: Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own., Conclusions: Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Somatostatin participants in multiple physiological functions by activating the five distinct G-protein-coupled receptors (sst1-sst5). In this study, we investigated the effect of sst5 activation on outward K currents in acutely isolated rat retinal ganglion cells using whole-cell patch-clamp techniques. Extracellular application of L-817,818, a specific sst5 agonist, significantly reduced outward K currents which was mainly the 4-aminopyridine and glybenclamide sensitive current components, but not the tetraethylammonium-sensitive one. The L-817,818 effect was mediated by sst5 since the suppression was eliminated when intracellular dialysis of the G-protein inhibitor GDP-β-S or extracellular application of the sst5 antagonist BIM-23056. Intracellular phospholipase C/protein kinase C signaling pathway was involved in the L-817,818 effect because the L-817,818 effect on K currents was inhibited when rat retinal ganglion cells were pretreated with U73122 or chelerythrine chloride. However, L-817,818 persisted to reduce the K currents when cAMP/protein kinase A, calcium/calmodulin-dependent protein kinase II and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathways were blocked respectively. These results suggest that sst5 activation suppresses 4-aminopyridine and glybenclamide-sensitive K currents in rat retinal ganglion cells by stimulating intracellular phospholipase C/protein kinase C signaling pathway, thereby regulating the rat retinal ganglion cell excitability.

Purpose: Corneal endothelial cell regeneration varies by species, with nonhuman primates (NHPs) and rabbits displaying low and high proliferative capacities, respectively. Recent studies report that topical application of rho-associated kinase (ROCK) inhibitors accelerates corneal endothelial wound healing in animal models and human patients with endothelial dysfunction. This study determines the regenerative capacity of canine corneal endothelial cells in vivo and their response to a topical ROCK inhibitor, Y27632, after transcorneal freezing., Methods: Right eyes of 6 beagles underwent transcorneal freezing; 10 mM ROCK inhibitor Y27632 or vehicle control was applied topically to both eyes at least 4 times daily for 56 days. Endothelial cell density was evaluated by in vivo confocal microscopy, and corneal thickness was measured by Fourier-domain optical coherence tomography (FD-OCT) and ultrasound pachymetry., Results: Transcorneal freezing induced severe central corneal edema in dogs, with restoration of transparency occurring within 4 weeks. Y27632 significantly decreased corneal thickness by FD-OCT and ultrasound pachymetry in the acute phase and significantly increased endothelial cell density at days 28 and 42 post-cryoinjury, suggesting faster restoration of endothelial cell recovery., Conclusions: Canine corneal endothelial function recovers at a similar rate as NHPs but more slowly than rabbits after cryoinjury. Faster corneal endothelial wound healing was observed by in vivo confocal microscopy and FD-OCT in dogs treated with Y27632 versus vehicle controls. Thus, a canine cryoinjury model may be a useful alternative to NHPs in detecting a response to therapies directed at endothelial regeneration.

Background and Purpose- Heme and iron are considered to be key factors responsible for secondary insults after intracerebral hemorrhage (ICH). Our previous study showed that LRP1 (low-density lipoprotein receptor-related protein-1)-Hx (hemopexin) facilitates removal of heme. The TLR7 (Toll-like receptor 7)-BTK (Bruton tyrosine kinase)-CRT (calreticulin) pathway regulates the expression of LRP1-Hx. This study is designed to clarify whether TLR7 activation facilitates heme scavenging and to establish the potential role of the BTK-CRT-LRP1-Hx signaling pathway in the pathophysiology of ICH. Methods- ICH was induced by stereotactic, intrastriatal injection of type VII collagenase. Mice received TLR7 agonist (imiquimod) via intraperitoneal injection after ICH induction. TLR7 inhibitor (ODN2088), BTK inhibitor (LFM-A13), and CRT agonist (thapsigargin) were given in different groups to further evaluate the underlying pathway. Mice were randomly divided into sham, ICH+vehicle (normal saline), ICH+Imiquimod (2.5, 5, and 10 μg/g), ICH+ODN2088, ICH+LFM-A13, ICH+thapsigargin, and ICH+ODN2088+thapsigargin. Imiquimod was administered twice daily starting at 6 hours after ICH; ODN2088 was administered by intracerebroventricular injection at 30 minutes, and LFM-A13 or thapsigargin was administered by intraperitoneal injection at 3 hours after ICH induction. Neurological scores, cognitive abilities, as well as brain edema, blood-brain barrier permeability, hemoglobin level, brain expression of TLR7/BTK/CRT/LRP1/Hx were analyzed. Results- Low dosage imiquimod significantly attenuated hematoma volume, brain edema, BBB permeability, and neurological deficits after ICH. Imiquimod also increased protein expressions of TLR7, BTK, CRT, LRP1, and Hx; ODN2088 reduced TLR7, BTK, CRT, LRP1, and Hx expressions. Conclusions- TLR7 plays an important role in heme scavenging after ICH by modulating the BTK-CRT-LRP1-Hx pathway. TLR7 may offer protective effects by promoting heme resolution and reduction of brain edema after ICH.

Background: Hepatocellular carcinoma (HCC) is an aggressive cancer with limited therapeutic options. Retrospective studies have shown that the administration of local anesthetics (LAs) during cancer surgery could reduce cancer recurrence. Besides, experimental studies reported that LAs could inhibit the growth of cancer cells. Thus, the purpose of this study was to investigate the effects of LAs on human HCC cells., Methods: The effects of 2 LAs (lidocaine and ropivacaine) (10 to 10 M) were studied after an incubation of 48 hours on 2 HCC cell lines, namely HuH7 and HepaRG. Cell viability, cell cycle analysis, and apoptosis and senescence tests were performed together with unsupervised genome-wide expression profiling and quantitative real-time polymerase chain reaction for relevant genes., Results: We showed that LAs decreased viability and proliferation of HuH7 cells (from 92% [P < .001] at 5 × 10 M to 40% [P = .02] at 10 M with ropivacaine and from 87% [P < .001] to 37% [P = .02] with lidocaine) and HepaRG progenitor cells (from 58% at 5 × 10 M [P < .001] to 29% at 10 M [P = .04] with lidocaine and 59% [P < .001] with ropivacaine 5 × 10 M) in concentration-dependent manner. LAs have no effect on well-differentiated HepaRG. Ropivacaine decreased the mRNA level of key cell cycle regulators, namely cyclin A2, cyclin B1, cyclin B2, and cyclin-dependent kinase 1, and the expression of the nuclear marker of cell proliferation MKI67. Lidocaine had no specific effect on cell cycle but increased by 10× the mRNA level of adenomatous polyposis coli (P < .01), which acts as an antagonist of the Wnt/β-catenin pathway. Both LAs increased apoptosis in Huh7 and HepaRG progenitor cells (P < .01)., Conclusions: The data demonstrate that LAs induced profound modifications in gene expression profiles of tumor cells, including modulations in the expression of cell cycle-related genes that result in a cytostatic effect and induction of apoptosis.

Background: Studies have reported that prophylactic continuous infusion of phenylephrine during spinal anesthesia for cesarean section can decrease the spread of local anesthetics. We investigated the ED50 of intrathecal hyperbaric ropivacaine in parturient women undergoing cesarean section under prophylactic infusion of phenylephrine., Methods: Sixty parturient women were allocated into 2 groups in this prospective study. Group P received 0.5 mL kg h of phenylephrine (5 mg/50 mL) at the start of intrathecal injection, and Group C (control group) received the same volume of saline. The dose of intrathecal ropivacaine for each subject was decided through up-down allocation method. The initial dose was set as 7.5 mg. Successful anesthesia was defined as the level of T6 or above achieved within 15 minutes after intrathecal injection and no additional epidural drug or venous analgesia to complete operation. The Massey formula was applied to calculate the ED50 of intrathecal ropivacaine in both groups., Results: The ED50 of hyperbaric ropivacaine determined by up-and-down method was 7.2 mg (95% confidence interval (CI), 6.8-7.6 mg) in the Group P, and 6.8 mg (95% CI, 6.4-7.2 mg) in the Group C, there was significant difference between the 2 groups (P < .5). The ED50 of intrathecal ropivacaine increases compared with Group C when phenylephrine is prophylactic infused to prevent spinal induced hypotension in cesarean section., Conclusion: The ED50 of intrathecal hyperbaric ropivacaine is 7.2 mg when phenylephrine is prophylactic infused to prevent spinal induced hypotension in cesarean section, and more ropivacaine demands on spinal anesthesia for cesarean section (www.chictr.org.cn, registration number: ChiCTR-RIC-17011650).

Purpose: To assess the short-term effects of instilling Y-27632, an inhibitor of Rho/Rho-associated protein kinases, on the chromatin supraorganization and DNA amount of corneal and limbal epithelial cells of healthy rats., Methods: Longitudinal sections (7 μm) of enucleated eyes of healthy rats that received, by instillation, balanced salt solution with or without 10 mM of Y-27632 daily for 7 or 15 days, were subjected to the Feulgen reaction. Feulgen-stained nuclei of corneal and limbal epithelial cells were studied by microscopy and video image analysis to establish the nuclear size (area and perimeter), supraorganization of chromatin (texture and degrees of condensation), and the Feulgen-DNA amount., Results: Instillation of Y-27632 for up to 15 days did not change the size of the nucleus or the chromatin texture of corneal and limbal epithelial cells. Samples treated with Y-27632 for 7 days showed condensed chromatin and a high Feulgen-DNA amount. Both corneal and limbal epithelium showed the presence of near-tetraploid nuclei corresponding to cells in the S and G2 phases of the cell cycle. The degrees of condensation and Feulgen-DNA amount of the nuclei of epithelial cells of the cornea and limbus of eyes from rats receiving Y-27632 for 15 days did not differ from control (no drug)., Conclusions: Changes in chromatin supraorganization and DNA amount, such as seen in this study, are indicative of cell proliferation and do not seem to be associated with disturbances in gene activity and transcription of DNA.

Background: Dexmedetomidine used as an adjuvant to local anesthetics may prolong the duration of peripheral nerve blocks. Whether this is mediated by a perineural or systemic mechanism remains unknown. The authors hypothesized that dexmedetomidine has a peripheral mechanism of action., Methods: The authors conducted a randomized, paired, triple-blind trial in healthy volunteers. All received bilateral saphenous nerve blocks with 20 ml ropivacaine, 0.5%, plus 1 ml dexmedetomidine, 100 µg/ml, in one thigh and 20 ml ropivacaine 0.5% plus 1 ml saline in the other thigh. The primary outcome measure was the duration of block assessed by temperature sensation (alcohol swab). The secondary outcome measure was the duration of block assessed by pinprick, pain during tonic heat stimulation, warmth detection threshold, and heat pain detection threshold., Results: All 21 enrolled volunteers completed the trial. The mean duration of block assessed by temperature sensation in the leg receiving ropivacaine plus dexmedetomidine was 22 h (95% CI, 21 to 24) compared to 20 h (95% CI, 19 to 21) in the leg receiving ropivacaine plus placebo with a mean difference of 2 h (95% CI, 1 to 3; P = 0.001). The duration of block was also significantly longer in the leg receiving dexmedetomidine when assessed by pinprick, pain during tonic heat stimulation, and warmth detection threshold but not heat pain detection threshold. One participant experienced numbness in an area in the leg receiving dexmedetomidine., Conclusions: Dexmedetomidine prolongs the duration of a saphenous nerve block by a peripheral mechanism when controlling for systemic effects but not necessarily to a clinically relevant extent.

Background: Rapid intravenous administration of lipid emulsion has become the standard treatment of severe local anesthetic systemic toxicity. This experiment in volunteers aimed at determining the effect of Intralipid® administration on the time to neurologic symptoms., Methods: Ropivacaine or levobupivacaine was infused intravenously in 16 volunteers (8 mg/min up to 120 mg) with 120 ml Intralipid® 20% (Fresenius, Paris France) or placebo infused at T + 2 min). Each subject received all four treatments in a crossover manner. The infusion was stopped after the intended dose had been administered or on occurrence of incipient neurologic signs of toxicity. The primary outcome was time-to-event. In addition, blood ropivacaine and levobupivacaine concentrations were measured., Results: The dose infused was not different whether volunteers received placebo (81.7 ± 22.3 vs. 80.8 ± 31.7 mg, ropivacaine vs. levobupivacaine) or Intralipid® (75.7 ± 29.1 vs. 69.4 ± 26.2 mg, ropivacaine vs. levobupivacaine), P = 0.755, Intralipid® versus placebo groups. Plasma concentrations were best modeled with an additional volume of distribution associated with Intralipid®. Simulations suggested that decreased peak concentrations would be seen if Intralipid® was given during a period of increasing concentrations after extravascular administration., Conclusions: At modestly toxic doses of ropivacaine or levobupivacaine, we were unable to find any effect of the infusion of Intralipid® on the time to early signs of neurologic toxicity in volunteers. Peak concentration was decreased by 26 to 30% in the subjects receiving Intralipid®. Simulations showed that Intralipid® might prevent the rapid increase of local anesthetic concentration after extravascular administration.

Because of its rapid onset time, recent years have seen an increase in the use of ultrasound (US)-guided popliteal sciatic nerve block (PSNB) via subparaneural injection for induction of surgical anesthesia. Moreover, in below-knee surgery, combined blocks, as opposed to sciatic nerve block alone, have become more common. These combined blocks often require a large volume of local anesthetic (LA), thus increasing the risk of local-anesthetic systemic toxicity (LAST). Thus, to decrease the risk of LAST, it is important to know the minimum effective volume (MEV) required for an adequate block. We, therefore, aimed to determine the MEV of ropivacaine 0.75% for induction of surgical anesthesia by the method of US-guided popliteal sciatic nerve block via subparaneural injection.Thirty patients underwent a US-guided PSNB with ropivacaine 0.75% at a 20-mL starting volume. Using a step-up/step-down method, we determined injection volumes for consecutive patients from the preceding patient's outcome. When an effective block was achieved within 40 minutes after injection, the next patient's volume was decreased by 2 mL. If the block failed, the next patient's volume was increased by 2 mL. The sensory and motor blockade was graded according to a 4-point scale. The block was considered a success if a combination of anesthesia and paresis (a score of 3 for both the sensory and motor nerves) was achieved within 40 minutes. The primary outcome measure was the MEV resulting in a successful subparaneural block of the sciatic nerve in 50% of patients (MEV50). Additionally, the data were processed with a probit regression analysis to determine the volume required to produce a complete sciatic nerve block in 90% of subjects (ED90).The MEV50 of 0.75% ropivacaine is 6.14 mL (95% confidence interval, 4.33-7.94 mL). The ED90 by probit analysis for a subparaneural injection was 8.9 mL (95% CI, 7.09-21.75 mL).The 6.14-mL MEV50 of ropivacaine 0.75% represents a 71% reduction in volume compared with neurostimulation techniques and a 14.7% reduction in volume compared with US-guided PSNB using the alternative perineural injection technique.

Objective: To evaluate the effect of 2% lidocaine, 0.5% bupivacaine, and 0.75% ropivacaine on the release of substance P (SP) and calcitonin gene-related peptide (CGRP) in skin wounds., Design: A primary, experimental, analytical, prospective, self-controlled, blinded study., Setting: The study is set in a university research center., Interventions: Twenty-eight Wistar rats were randomly divided into 4 groups: lidocaine, bupivacaine, ropivacaine, and the control. After general anesthesia, a local anesthetic or 0.9% saline (control) was injected subdermally along a 2-cm line on the dorsal midline of each rat; 30 minutes later, an incision (nociceptive stimulus) was made along this line. The animals were euthanized, and skin samples were collected from the center of the incision line and sent for CGRP and SP quantification., Main Outcome Measures: Quantification of CGRP and SP by Western blotting., Results: Substance P levels were similar in the lidocaine and ropivacaine groups but were significantly lower than those of the control group (P = .002); no significant difference in SP levels was found between the bupivacaine and control groups. Procalcitonin gene-related peptide levels were significantly lower in the experimental groups than those in control subjects (P = .009), with no significant differences among the experimental groups. No significant differences in CGRP levels were found among all groups. Lidocaine and ropivacaine inhibited SP release. All 3 local anesthetics inhibited the release of procalcitonin gene-related peptide, but not the release of CGRP in rat skin., Conclusions: Lidocaine and ropivacaine may inhibit neurogenic inflammation by biochemical pathways activated by SP, whereas bupivacaine seems to have no influence on this process.

Study Design: Establishment of immortalized cell lines derived from rat intervertebral disc cells by Rho-associated kinase (ROCK) inhibitor, Y-27632., Objective: To determine whether rat nucleus pulposus (NP) and annulus fibrosus (AF) cells could be immortalized, retain their phenotype, and used as cell lines for in vitro cell biology., Summary of Background Data: Intervertebral disc degeneration is a major factor for most low-back pain. However, the mechanism of the disease is not well understood by the limitation to obtain sufficient amounts of primary disc cells. Therefore, the establishment of disc cell lines will help in vitro molecular signaling studies to understand the mechanism of degenerative disc disease., Methods: Cells were isolated from the NP and AF tissues of lumbar discs of adult Sprague Dawley rat. Tissues were digested and cultured with DMEM/Ham's F-12 (1:1) and 20% FBS and antibiotics. From day 3, cells were grown in the presence of 10  μM Y-27632, a well-characterized inhibitor of the ROCK, and subcultured by trypsinization, passaging them 1:3 onto 100  mm culture dishes. Morphologic and genetic analyses were performed on the different passaged cells., Results: ROCK inhibitor successfully immortalized rat NP and AF cells. They passaged for over 50 generations with sustained expression levels of several NP and AF cell markers. In addition, they retained phenotypic features similar to the primary parental NP and AF cells when the cells were challenged with different cytokines and growth factors., Conclusion: We established immortalized rat NP and AF cell lines using a method of treating cells with ROCK inhibitor Y-27632 and demonstrated that these immortalized cells retain the properties of primary cells and could serve as useful tools for in vitro signaling studies or drug screening studies to develop novel therapeutic strategies., Level of Evidence: N/A.

Objective: This study determined whether angiotensinogen (AGT) has angiotensin II-independent effects using multiple genetic and pharmacological manipulations., Approach and Results: All study mice were in low-density lipoprotein receptor -/- background and fed a saturated fat-enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were >90% lower compared with their wild-type littermates. HypoAGT mice had lower systolic blood pressure, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in low-density lipoprotein receptor -/- mice. In mice with established adiposity, administration of AGT antisense oligonucleotide versus aliskiren led to equivalent reductions of systolic blood pressure and atherosclerosis. AGT antisense oligonucleotide administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat-enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat, and human revealed 4 highly conserved sequences within the des(angiotensin I)AGT domain. des(angiotensin I)AGT, through adeno-associated viral infection in hepatocyte-specific AGT-deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis., Conclusions: AGT contributes to body weight gain and liver steatosis through functions of the des(angiotensin I)AGT domain, which are independent of angiotensin II production., (© 2015 American Heart Association, Inc.)

Background: Tacrolimus (Tac) and Cyclosporine A (CyA) calcineurin inhibitors (CNIs) are 2 effective immunosuppressants which are essential to prevent allograft rejection. Calcineurin inhibitors are known to be nephrotoxic. However, the precise mechanism of nephrotoxicity is not fully understood. In this study, we investigated the in vivo effects of CNIs on the local renal renin-angiotensin system in the collecting duct (CD)., Methods: Three-week-old mice were treated with either vehicle, CyA (2 mg/kg per day), Tac (0.075 mg/kg per day), CyA + Aliskiren (25 mg/kg per day), or Tac + Aliskiren for 3 weeks. Serum creatinine was measured. Renin and vascular endothelial growth factor (VEGF) contents in CD were evaluated with flow cytometry and multiphoton microscopy. The diameter of vessels was assessed with multiphoton microscopy, and the amount of renal collagen was determined by real-time polymerase chain reaction and Masson staining., Results: The elevated level of serum creatinine in CNI groups was abolished by Aliskiren. Flow cytometric analysis found elevated renin content in principal cells, which was prevented by Aliskiren. This result was further confirmed with multiphoton microscopy. The VEGF content in CD correlated with reduced capillary diameter and with the formation of fibrotic islands., Conclusions: Calcineurin inhibitors induce production of renin in the CD that may contribute to decreased renal blood flow. In turn, CD responds with increased VEGF production, resulting in disproportional vessel growth, further worsening the local hypoxia and striped fibrosis surrounding the CDs. Aliskiren, a direct renin inhibitor blocks these effects and improves CNI-induced nephropathy by decreasing renin production in the CDs. Our data suggest that Aliskiren may be used for the prevention of CNI nephrotoxicity.

Background: Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning., Methods and Results: Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis., Conclusions: Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis., (© 2015 American Heart Association, Inc.)

Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.

Background: Even though ropivacaine is frequently used during orthopedic surgery, the relationship between plasma concentrations and degree of sensory anesthesia after a peripheral nerve block is currently unknown. The aim of this study was to characterize this relation using population pharmacokinetic-pharmacodynamic modeling., Methods: Femoral nerve block was performed by the anterior approach using a single injection (20 ml) of 0.5% ropivacaine hydrochloride in 20 patients scheduled for total knee arthroplasty under spinal anesthesia. Sensory thresholds in response to a gradual increase in transcutaneous electrical stimulation (primary endpoints), loss and recovery of ice-cold sensation, as well as total ropivacaine plasma concentrations were determined up to 4 days after administration of the local anesthetic. Using NONMEM (ICON, USA), sensory block was modeled by assuming an equilibration delay (ke0) between amount in the depot and effect-site compartments., Results: Mean effect-site amount producing 90% of the maximum possible effect (AE90) was estimated as 20.2 mg. At 2 × AE90, the sigmoid Emax model predicted a mean onset time of 23.4 min and mean duration of 22.9 h. Interindividual variability (IIV) for AE50 was 49%. Typical ke0 half-life was 34.7 min (IIV = 52%) and steepness parameter 8.7 (IIV = 48%). None of the pharmacodynamic model parameters showed sex, age, or body weight dependency., Conclusions: A population pharmacokinetic/pharmacodynamic model was developed that quantitatively describes the sensory component of a femoral nerve block in orthopedic patients. Further clinical studies will be needed to validate the clinical relevance of this finding.

Phospholipase A2 (PLA2) changes the phosphatidylcholine contained in low-density lipoprotein (LDL) to lysophosphatidylcholine (LPC), which has various proatherogenic properties. We reported that tumor necrosis factor-alpha (TNFα) enhanced the expression of group V PLA2 (sPLA2-V) in human umbilical vein endothelial cells (HUVECs), and the LPC content in LDL and the monocyte chemoattractant protein-1 (MCP-1) expression were augmented when TNFα-stimulated HUVECs were incubated with LDL. Here, we observed that an HMG-CoA reductase inhibitor, pitavastatin, at the concentration of >1 μM administered 12 hours before TNFα stimulation suppressed the enhancement of sPLA2-V mRNA and protein. Pitavastatin also prevented the enhancement of the LPC content in LDL and the expression of MCP-1 mRNA when TNFα-stimulated HUVECs were incubated with LDL. The administration of geranylgeranyl pyrophosphate restored the expression of sPLA2-V mRNA and protein. The administration of the Rho kinase inhibitor Y-27632 and the transfection of small interfering RNA (siRNA) against sPLA2-V before TNFα stimulation both diminished the TNFα-induced sPLA2-V mRNA expression. Therefore, Y-27632 and siRNA against sPLA2-V also prevented the enhancement of MCP-1 mRNA expression when TNFα-stimulated HUVECs were incubated with LDL. Pitavastatin's inhibitory effect on the expression of sPLA2-V induced by TNFα may be useful to prevent the proatherogenic modification of LDL.

Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal endothelial transplantation. However, despite the value and potential of endothelial graft surgery, a strictly pharmacological approach for treating corneal endothelial dysfunction remains an attractive proposition. Previously, we reported that the selective Rho-associated kinase (ROCK) inhibitor Y-27632 promotes cell adhesion and proliferation, and inhibits the apoptosis of primate corneal endothelial cells in culture. These findings have led us to develop a novel medical treatment for the early phase of corneal endothelial disease using ROCK inhibitor eye drops. In rabbit and monkey models of partial endothelial dysfunction, we showed that corneal endothelial wound healing was accelerated via the topical application of ROCK inhibitor to the ocular surface, resulting in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. Based on these animal studies, we are now attempting to advance the clinical application of ROCK inhibitor eye drops for patients with corneal endothelial dysfunction. A pilot clinical study was performed at the Kyoto Prefectural University of Medicine, and the effects of Y-27632 eye drops after transcorneal freezing were evaluated in 8 patients with corneal endothelial dysfunction. We observed a positive effect of ROCK inhibitor eye drops in treating patients with central edema caused by Fuchs corneal endothelial dystrophy. We believe that our new findings will contribute to the establishment of a new approach for the treatment of corneal endothelial dysfunction.

Background: Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase-Akt-nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability., Methods: Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed., Results: Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10 M for ropivacaine; IC50 = 5.864 × 10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10 M for ropivacaine; IC50 = 6.377 × 10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated., Conclusions: Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory "side-effect" of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.

Rho kinase (ROCK) is a major downstream effector of the small GTPase RhoA. ROCK family, consisting of ROCK1 and ROCK2, plays central roles in the organization of actin cytoskeleton and is involved in a wide range of fundamental cellular functions, such as contraction, adhesion, migration, proliferation, and apoptosis. Due to the discovery of effective inhibitors, such as fasudil and Y27632, the biological roles of ROCK have been extensively explored with particular attention on the cardiovascular system. In many preclinical models of cardiovascular diseases, including vasospasm, arteriosclerosis, hypertension, pulmonary hypertension, stroke, ischemia-reperfusion injury, and heart failure, ROCK inhibitors have shown a remarkable efficacy in reducing vascular smooth muscle cell hypercontraction, endothelial dysfunction, inflammatory cell recruitment, vascular remodeling, and cardiac remodeling. Moreover, fasudil has been used in the clinical trials of several cardiovascular diseases. The continuing utilization of available pharmacological inhibitors and the development of more potent or isoform-selective inhibitors in ROCK signaling research and in treating human diseases are escalating. In this review, we discuss the recent molecular, cellular, animal, and clinical studies with a focus on the current understanding of ROCK signaling in cardiovascular physiology and diseases. We particularly note that emerging evidence suggests that selective targeting ROCK isoform based on the disease pathophysiology may represent a novel therapeutic approach for the disease treatment including cardiovascular diseases.

Purpose of Review: Spinal anesthesia (SPA) has not been popular for day-case surgery because of prolonged neurologic blockade with long-acting local anesthetics such as bupivacaine, thereby delaying discharge. Although the intermediate duration of action of lidocaine and mepivacaine appears to be more suitable for day-case surgery, their use is not deemed appropriate by many because of a high incidence of transient neurologic symptoms (TNSs). The present review summarizes recent clinical data on the intrathecal use of alternative local anesthetics and adjuvants that may offer valuable alternatives to general anesthesia in day-case surgery., Recent Findings: Prilocaine has a similar intrathecal pharmacokinetic profile as lidocaine but with a significantly lower risk of TNSs. Onset of spinal after 2-chloroprocaine is comparable with lidocaine or prilocaine, but with a considerably shorter duration of action. Also, TNS is clearly less frequent compared with lidocaine. Although its intrathecal use has recently been approved in Europe, this is still considered to be off-label in the USA. Articaine provides an extraordinary fast onset and a short duration of spinal block, the latter being approximately intermediate between chloroprocaine and prilocaine. However, articaine is associated with a high risk for intraoperative hypotension and a small risk for TNS, albeit but less frequent than after lidocaine. Concerns regarding possible neurotoxicity of articaine remain to be resolved., Summary: SPA for day cases might become a most valuable method for ambulatory surgery when using short acting local anesthetics. This, however, not only depends on drugs being used but also on infrastructure (post anaesthesia care unit) and organizational issues.

Calcium desensitization plays a critical role in the occurrence of vascular hyporeactivity after shock. Interleukin (IL)-1β participates in the regulation of vascular reactivity via both nitric oxide (NO)-dependent and NO-independent mechanisms. However, the specific NO-independent pathway remains to be established. The issue of whether IL-1β modulates vascular reactivity via regulation of calcium sensitivity in the NO-independent mechanism is unclear. In the current study, effects of IL-1β on vascular calcium sensitivity and its relationship with PKC and Rho kinase were investigated in vivo and in vitro using a rabbit model of lipopolysaccharide (LPS)-induced endotoxic shock and superior mesenteric arteries (SMAs), respectively. The calcium sensitivity profile of SMAs displayed a biphasic change after LPS-induced endotoxic shock (significant increase at 0.5 hour and 1 hour after LPS administration and marked decrease after 2 hours) and was negatively related to changes in serum IL-1β. The IL-1 receptor antagonist, IL-1ra (4 μg/mL), partly reversed LPS-induced calcium desensitization. In vitro incubation with IL-1β (50-200 ng/mL) reduced the calcium sensitivity of SMAs and suppressed the activities of Rho kinase and PKC and the phosphorylation of 20-kDa myosin light chain. These effects of IL-1β were shown to be regulated by the PKC agonist, phorbol 12-myristate 13-acetate, and Rho kinase agonist and antagonist, angiotensin II, and Y-27632, respectively. Our results collectively suggest that IL-1β participates in vascular hyporeactivity after endotoxic shock via regulation of vascular calcium sensitivity. Moreover, this regulatory effect of IL-1β seems closely related to downregulation of the activities of PKC and Rho kinase.

Objective: This study aimed to investigate the effect of serum taken from patients with severe acute pancreatitis (SAP) on vascular endothelial permeability., Methods: The monolayer permeability of endothelial cells (ECs) was assessed. Morphological changes in ECs, induced by serum from patients with SAP were assessed. Expressions of RhoA, myosin light chain (MLC) phosphorylation, and VE-cadherin protein were detected by Western blot., Results: Compared with the control group, 20% SAP serum significantly increased endothelial monolayer permeability (P < 0.01), markedly induced transcellular F-actin redistribution with stress fiber formation and VE-cadherin derangement with fragmentations located at the cell borders, and increased gaps between ECs. Furthermore, Western blotting showed that SAP serum induced rapid activation of Rho protein, and markedly increased the level of phosphorylated MLC. However, pretreatment with Y-27632 (an inhibitor for Rho kinase) significantly inhibited endothelial hyperpermeability and the morphological changes of F-actin rearrangement and VE-cadherin redistribution. This was associated with a down-regulation of Rho protein expression and a reduction in the level of MLC phosphorylation., Conclusions: The SAP serum induces the loss of vascular endothelial monolayer integrity, with endothelial F-actin stress fiber formation and VE-cadherin redistribution. One of the mechanisms for this process involves the activation of the Rho/Rho kinase signaling pathway.

Objectives: We examined the mechanism of coronary artery spasm related to oxidant stress with aging in senescence marker protein-30 (SMP30)-deficient mice because SMP30 decreases with aging and SMP30 knockout (KO) mice show a short life with increased oxidant stress., Methods: To examine the effect of SMP30 on coronary artery vasomotor tone, we measured the endothelium-dependent [5-hydroxytryptamine (5-HT)] response of isolated, pressurized coronary arteries from SMP30 KO and wild-type (WT) mice (n=10 each)., Results: In SMP30 KO mice, 5-HT-induced vasoconstriction occurred, which altered vasodilation with dithiothreitol, a thiol-reducing agent. In WT mice, 5-HT-induced vasodilation occurred. Administration of 5-HT from the aortic sinus induced a coronary artery spasm in SMP30 KO mice, which was prevented by the intravenous administration of Y-27632, rho-kinase inhibitor. The fluorescence level of monochlorobimane in coronary arteries, which covalently labels the reduced total thiols, decreased in SMP30 KO mice, but reverted to a level comparable with that of WT mice on treatment with Y-27632. From these results, SMP30 provides protection against coronary artery spasm., Conclusion: Chronic oxidant stress associated with aging plays an important role in coronary artery spasm related to thiol oxidation and rho-kinase signaling.

Objective: A nonsynonymous single nucleotide polymorphism (SNP) in the SLCO2B1 gene encoding organic anion transporting polypeptide 2B1 (OATP2B1), c.935G>A (p.R312Q; rs12422149), has been associated with reduced plasma concentrations of montelukast in patients with asthma. Our aim was to examine the possible effects of the SLCO2B1 c.935G>A SNP on the single-dose pharmacokinetics of the suggested OATP2B1 substrates montelukast and aliskiren., Methods: Sixteen healthy volunteers with the SLCO2B1 c.935GG genotype, 12 with the c.935GA genotype, and five with the c.935AA genotype ingested a single 10 mg dose of montelukast or a 150 mg dose of aliskiren, with a washout period of 1 week. Plasma montelukast concentrations were measured up to 24 h. Plasma and urine aliskiren concentrations were measured up to 72 and 12 h, respectively, and plasma renin activity up to 24 h after aliskiren intake., Results: The SLCO2B1 genotypes had no significant effect on the pharmacokinetics of montelukast or aliskiren. The geometric mean ratios with 90% confidence intervals of montelukast area under the plasma concentration-time curve from 0 h to infinity (AUC(0-∞)) in participants with the c.935GA or the c.935AA genotype to those with the c.935GG genotype were 1.02 (0.87, 1.21) or 0.88 (0.71, 1.10), respectively (P=0.557). The geometric mean ratios (90% confidence interval) of aliskiren AUC(0-∞) in participants with the c.935GA or the c.935AA genotype to those with the c.935GG genotype were 0.98 (0.74, 1.30) or 1.24 (0.85, 1.80), respectively (P=0.576)., Conclusion: These data do not support the suggested functional significance of the SLCO2B1 c.935G>A SNP on OATP2B1 activity in vivo., (© 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.)

Aldosterone is increased in diabetes and contributes to the development of diabetic nephropathy. The authors hypothesized that reduction in aldosterone production in diabetes by amlodipine or aliskiren improves diabetic kidney disease by attenuating renal oxidative stress and fibrosis. Normoglycemic and streptozotocin-induced diabetic Sprague-Dawley rats were given vehicle, amlodipine, or aliskiren alone and combined for 6 weeks. At the end of study, we evaluated blood pressure (BP), 24-hour urinary sodium (UNaV) and aldosterone excretion rates, renal interstitial fluid (RIF) levels of nitric oxide (NO), cyclic guanosine 3',5'-monophosphate (cGMP), and 8-isoprostane, and renal morphology. BP was not significantly different between any of experimental groups. UNaV increased in diabetic animals and was not affected by different treatments. Urinary aldosterone excretion increased in diabetic rats receiving vehicle and decreased with amlodipine and aliskiren alone or combined. RIF NO and cGMP levels were reduced in vehicle-treated diabetic rats and increased with amlodipine or aliskiren given alone and combined. RIF 8-isoprostane levels and renal immunostaining for periodic acid-Schiff and fibronectin were increased in vehicle-treated diabetic rats and decreased with aliskiren alone or combined with amlodipine. The authors conclude that inhibition of aldosterone by amlodipine or aliskiren ameliorates diabetes induced renal injury via improvement of NO-cGMP pathway and reduction in oxidative stress and fibrosis, independent of BP changes.

Background: Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have antiinflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells., Methods: NCI-H838 lung cancer cells were incubated with tumor necrosis factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1 nM-100 μM). Cell migration and total cell lysate Src-activation and intercellular adhesion molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated., Results: Ropivacaine treatment (100 μM) of H838 cells for 20 min decreased basal Src activity by 62% (P=0.003), and both ropivacaine and lidocaine coadministered with tumor necrosis factor-α statistically significantly decreased Src-activation and intercellular adhesion molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 h was inhibited by 26% (P=0.005) in presence of 1 μM ropivacaine and 21% by 1 μM lidocaine (P=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition., Conclusions: This study indicates that amide-, but not ester-linked, local anesthetics may provide beneficial antimetastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of tumor necrosis factor-α-induced Src-activation and intercellular adhesion molecule-1 phosphorylation, providing the first evidence of a molecular mechanism that appears to be independent of their known role as sodium-channel blockers.

Objective: Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules and cells. Adherens junctions (AJ) play a central role in regulating this barrier. We aim to investigate the role of a distinctive 3-dimensional reticular network of AJ found in the endothelium., Methods and Results: In endothelial AJ, vascular endothelial-cadherin recruits the cytoplasmic proteins β-catenin and p120-catenin. β-catenin binds to α-catenin, which links AJ to actin filaments. AJ are usually described as linear structures along the actin-rich intercellular contacts. Here, we show that these AJ components can also be organized in reticular domains that contain low levels of actin. Reticular AJ are localized in areas where neighboring cells overlap and encompass the cell adhesion receptor platelet endothelial cell adhesion molecule-1 (PECAM-1). Superresolution microscopy revealed that PECAM-1 forms discrete structures distinct from and distributed along AJ, within the voids of reticular domains. Inflammatory tumor necrosis factor-α increases permeability by mechanisms that are independent of actomyosin-mediated tension and remain incompletely understood. Reticular AJ, but not actin-rich linear AJ, were disorganized by tumor necrosis factor-α. This correlated with PECAM-1 dispersal from cell borders. PECAM-1 inhibition with blocking antibodies or small interfering RNA specifically disrupted reticular AJ, leaving linear AJ intact. This disruption recapitulated typical tumor necrosis factor-α-induced alterations of barrier function, including increased β-catenin phosphorylation, without altering the actomyosin cytoskeleton., Conclusions: We propose that reticular AJ act coordinately with PECAM-1 to maintain endothelial barrier function in regions of low actomyosin-mediated tension. Selective disruption of reticular AJ contributes to permeability increase in response to tumor necrosis factor-α.

Objective: Sex differences in cerebral ischemic injury are, in part, attributable to the differences in cerebrovascular perfusion. We determined whether the brain microvascular endothelial cells (ECs) isolated from the female brain are more resistant to ischemic injury compared with male ECs, and whether the difference is attributable to lower expression of soluble epoxide hydrolase and higher levels of vasoprotective epoxyeicosatrienoic acids (EETs). We also determined whether protection by EETs is linked to the inhibition of rho-kinase (ROCK)., Methods and Results: EC ischemic damage was measured after oxygen-glucose deprivation (OGD) using propidium iodide (PI) and cleaved caspase-3 labeling. Expression of soluble epoxide hydrolase was determined by quantitative polymerase chain reaction and immunocytochemistry, EETs levels by liquid chromatography-tandem mass spectrometry, and ROCK activity by ELISA. EC damage was higher in males compared with females, which correlated with higher soluble epoxide hydrolase mRNA, stronger immunoreactivity, and lower EETs compared with female ECs. Inhibition of soluble epoxide hydrolase abolished the sex difference in EC damage. ROCK activity was higher in male versus female ECs after OGD, and sex differences in EC damage and ROCK activity were abolished by 14,15-EET and ROCK inhibition., Conclusions: Sex differences in ischemic brain injury are, in part, attributable to differences in EET-mediated inhibition of EC ROCK activation after ischemia.

Objective: Chronic activation of the renin-angiotensin-aldosterone system is a major contributing factor to the pathogenesis and progression of cardiovascular and renal diseases., Methods: To evaluate the role of renin-angiotensin-aldosterone system blockade with aliskiren, a direct renin inhibitor, in the development and progression of dilated cardiomyopathy in the Syrian cardiomyopathic hamster (SCH) model, we treated 1-month-old SCH with aliskiren (10 mg·kg·d) over a 4-month period. For comparative purposes, we also evaluated the effects of the angiotensin receptor blocker valsartan (10 mg·kg·d) and the combination of both drugs. Age-matched golden hamsters were used as controls. Left ventricular end-diastolic volume and end-systolic volume, ejection fraction, and diastolic function were determined by echocardiography. Systolic blood pressure (SBP) was also measured in the left femoral artery by sphygmomanometry., Results: Results indicate that at 2 months of age, SBP is higher in SCH than in controls, and administration for 1 month of aliskiren, valsartan, or the combination of these drugs normalized SBP in SCH to a similar extent. In 5-month-old SCH, aliskiren improved ejection fraction (from 48.6% ± 5.8% to 69.4% ± 3.2%, n = 5, P < 0.05), left ventricular end-systolic volume (from 0.28 ± 0.06 to 0.10 ± 0.01 mL/100 g body weight), left ventricular end-diastolic volume (from 0.61 ± 0.05 to 0.34 ± 0.02 mL/100 g body weight), and normalized diastolic function (E:A ratio increases from 0.93 ± 0.13 to 1.70 ± 0.03, n = 5, P < 0.05). Similar results were observed with valsartan or the combination of aliskiren and valsartan., Conclusions: Our results indicate that in this animal model, aliskiren is as effective as valsartan, or the combination of both drugs, in improving diastolic function and in preventing the development of dilated cardiomyopathy. These findings suggest that aliskiren may be used as a monotherapy in heart failure management. Clinical studies, however, are needed to assess the effectiveness of this drug in patients with heart failure.

The cerebrovascular remodeling is a prominent feature of hypertension and considered as a major risk of stroke. Statins may suppress the activation of the Rho/Rho-kinase pathway and have pleiotropic actions against the development of vascular remodeling. We hypothesized that the inhibition of the Rho/Rho-kinase pathway by simvastatin during hypertension could recuperate the pathological changes of basilar artery through the downregulation of cell proliferation. To resolve the problem, we used 2-kid, 2-clip rat as a hypertension model and evaluated the effect of simvastatin on the Rho/Rho-kinase pathway. In addition, we assessed the changes of the proliferation rate by CCK-8 assay in basilar artery smooth muscle cells. Our results from this study showed that a continuous increase in the plasma endothelin-1 (ET-1) concentration and the Rho/Rho-kinase activity was positively correlated with changes in blood pressure in the hypertensive rat. Simvastatin ameliorated the upregulated Rho/Rho-kinase activity and cell proliferation during hypertension. Moreover, simvastatin, the RhoA inhibitor C3, and the RhoA-kinase inhibitor Y27632 all attenuated the proliferation rate induced by ET-1 in basilar artery smooth muscle cells via the Rho/Rho-kinase signaling pathway. In conclusion, simvastatin attenuated ET-1-induced proliferation through the Rho/Rho-kinase signaling pathway in hypertensive rat basilar artery, and it may be an excellent reagent to protect vascular remodeling and stroke.

The development of sepsis is multifactorial. Tissue damage and organ dysfunction may be caused not only by the microorganisms but also by the inflammatory mediators released in response to the infection. Interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) levels in serum are well known to be upregulated in humans with sepsis and can be used to predict outcome. Using human umbilical vein endothelial cells, we analyzed the role of guanine-nucleotide exchange factor H1 (GEF-H1) on lipopolysaccharide (LPS)-dependent IL-6/TNF-α expression in endothelial cells. Lipopolysaccharide upregulated IL-6 secretion in a dose- and time-dependent manner. Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced nuclear factor κB (NF-κB) p65 phosphorylation, IL-6/TNF-α messenger RNA (mRNA), and IL-6/TNF-α protein productions. Guanine-nucleotide exchange factor H1 protein expression remained on a high level among 1 to 9 h in response to LPS challenge of endothelial cells. Inhibition of GEF-H1 by specific small interfering RNA or inactivation of Rho-associated kinase with Y-27632 not only significantly reduced LPS-induced p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) activities but also blocked LPS-induced NF-κB translocation and activation, thereby inhibiting IL-6/TNF-α mRNA and protein productions. Furthermore, SB203580 (p38 inhibitor) but not PD98059 (ERK1/2 inhibitor) blocked LPS-induced NF-κB activation; however, both inhibitors significantly suppressed IL-6/TNF-α mRNA and protein expression. In summary, our data suggest that LPS rapidly upregulates GEF-H1 expression. Activated Rho-associated kinase by GEF-H1 subsequently activates p38 and ERK1/2, thereby increasing IL-6/TNF-α expression in endothelial cells. P38 and ERK1/2 regulate LPS-induced IL-6/TNF-α expression through an NF-κB-dependent manner and an NF-κB-independent manner, respectively.

Treatment strategies to improve blood pressure control, reduce end-organ damage, and improve cardiovascular outcomes are more important today than ever before. Most patients will require combination therapy to achieve target blood pressure; early initiation of combination therapy may help patients achieve blood pressure control more rapidly. Low-dose combinations may be more effective with fewer adverse effects than higher doses of single agents. Dysregulation of the renin-angiotensin-aldosterone system (RAAS) is an important contributor in the pathogenesis of hypertension and its sequelae. Treatment with a direct renin inhibitor blocks the rate-limiting step in the RAAS, resulting in decreased angiotensin I and II production and decreased urinary aldosterone excretion. Like the angiotensin converting enzyme inhibitors and angiotensin II receptor blockers, treatment with a direct renin inhibitor increases plasma renin concentration, but unlike the other RAAS inhibitors, treatment with a direct renin inhibitor decreases plasma renin activity. This unique combination of effects on the RAAS make a direct renin inhibitor an attractive option to combine with other antihypertensive agents for the management of hypertension and its comorbidities. Clinical studies have shown that combining the direct renin inhibitor, aliskiren, with drugs representing each of the major classes of antihypertensive agents (thiazide diuretics, beta blockers, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, and calcium-channel blockers) reduces blood pressure, improves markers for cardiovascular outcomes, or does both. Results of several ongoing randomized clinical trials should provide additional insights into the potential of therapeutic combinations that include aliskiren to improve cardiovascular morbidity and mortality in patients with hypertension and related comorbidities.

Objectives: Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells., Methods: We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (5-chloro-1H-indole-2-carboxylic acid [1-(4-fuorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2 oxoethyl] amide) (25, 50, and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC (stable isotope labeling with amino acids in cell culture) method., Results: A total of 22 protein spots and 4 phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of mitogen-activated protein kinase/extracellular signal-regulated kinase and tumor necrosis factor α/nuclear factor κB., Conclusions: Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through 1 pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown, and posttranslational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways.

We hypothesized that compared with hydrochlorothiazide (HCTZ), the renin inhibitor aliskiren (ALISK) or amlodipine (AMLO) and their combination reduce albuminuria via reduction in renal inflammation, independent of blood pressure (BP) changes. We studied normal and streptozotocin-induced diabetic (DM) Sprague-Dawley rats treated for 6 weeks with vehicle, ALISK, HCTZ, or AMLO individually and combined and evaluated the effects of treatments on BP, urine albumin to creatinine ratio, renal interstitial fluid levels of angiotensin II, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) and renal expression of TNF-α, IL-6, transforming growth factor beta 1, and nuclear factor kappa B. There were no differences in BP between treatments. Only ALISK and its combinations reduced renal interstitial fluid angiotensin II. Urine albumin to creatinine ratio increased in DM rats and decreased with ALISK alone or combined with HCTZ or AMLO. HCTZ or AMLO individually and combined did not influence urine albumin to creatinine ratio. Renal interstitial fluid TNF-α and IL-6, and the renal expression of TNF-α, IL-6, transforming growth factor beta 1, and nuclear factor kappa B were increased in DM rats. These renal inflammatory markers were reduced only with ALISK or AMLO individually or combined with other treatments. We conclude that ALISK alone and combined with HCTZ or AMLO reduced albuminuria in diabetes via reduction in renal inflammation, independent of BP changes.

Excessive neutrophil infiltration is a major component in septic lung injury, although the signaling mechanisms behind pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. Herein, we hypothesized that Rho-kinase activity may play a significant role in pulmonary neutrophil recruitment and tissue damage in abdominal sepsis. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 (0.5 or 5 mg/kg) before cecal ligation and puncture (CLP). Bronchoalveolar lavage fluid and lung tissue were harvested for analysis of neutrophil infiltration, as well as edema and CXC chemokine formation. Blood was collected for analysis of Mac-1 on neutrophils and CD40L on platelets as well as soluble CD40L and matrix metalloproteinase 9 (MMP-9) in plasma. Cecal ligation and puncture triggered significant pulmonary damage characterized by neutrophil infiltration, increased levels of CXC chemokines, and edema formation in the lung. Furthermore, CLP upregulated Mac-1 expression on neutrophils, decreased CD40L on platelets, and increased soluble CD40L and MMP-9 in the circulation. Interestingly, inhibition of Rho-kinase dose-dependently decreased CLP-induced neutrophil expression of Mac-1, formation of CXC chemokines and edema, as well as neutrophil infiltration and tissue damage in the lung. Moreover, Rho-kinase inhibition significantly reduced sepsis-provoked gene expression of CXC chemokines in alveolar macrophages. In contrast, Rho-kinase inhibition had no effect on platelet shedding of CD40L or plasma levels of MMP-9 in septic mice. In conclusion, these data demonstrate that the Rho-kinase signaling pathway plays a key role in regulating pulmonary infiltration of neutrophils and tissue injury via regulation of CXC chemokine production in the lung and Mac-1 expression on neutrophils in abdominal sepsis.

Background: The chemokine receptors CCR5 and CXCR3 are expressed by T cells and macrophages. We examined effects of a CCR5/CXCR3 antagonist (TAK), with a particular focus on the role of macrophages, in a rat kidney transplant model., Methods: Dark Agouti rat kidneys were transplanted into Lewis rats. The recipients were treated daily with a 10 mg/kg TAK on posttransplant days 0 to 14 and/or 2 mg/kg of cyclosporine A (CsA) on days 0 to 5. Graft survival, histological changes, and the expression of chemokines and chemokine receptors on T cells and macrophages were studied., Results: Treatment with TAK alone suppressed CD4+T cell infiltration and slightly prolonged graft survival. The expressions of both CCR5 and CXCR3, and activated macrophage-associated cytokines and chemokines, were significantly increased on macrophages that had been separated from rejecting kidneys, compared with those from spleens. However, these upregulations were decreased in macrophages from kidneys that had been treated with TAK. Immunohistochemistry also showed that macrophages infiltrating tubules of rejecting kidney expressed both receptors. In the CsA alone group, macrophages were the dominant infiltrating cells, and all allografts were rejected within 10 days. A combined therapy involving CsA and TAK resulted in decreased macrophage infiltration, and graft survival was substantially prolonged. The levels of activated macrophage-associated cytokines and chemokines were also decreased., Conclusion: The dual blocking of CCR5/CXCR3 can be useful in decreasing rejection, with or without CsA. This mechanism acts, not only to block T-cell recruitment to a kidney graft but to suppress the infiltration of macrophages as well.

Background: Blockade of the renin-angiotensin system (RAS) with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor blockers (ARBs) slow the progression of various chronic kidney diseases and chronic allograft dysfunction. RAS inhibition can be achieved also by directly blocking renin upstream from ACE. However, direct renin inhibition can have additional effects since formation of renoprotective Ang II breakdown products such as angiotensin (Ang) (1-7) that are produced by ACE2 are also inhibited., Methods: Using a Fischer-to-Lewis renal transplantation model, the effect of the renin inhibitor aliskiren (10 mg/kg/day) was assessed on the development of chronic allograft dysfunction compared with vehicle treatment and Ang II receptor blockers candesartan., Results: Aliskiren had no effect on renal function (proteinuria, creatinine clearance) or on renal morphological changes (glomerulosclerosis collagen deposition, myofibroblast accumulation and macrophage infiltration) compared with the vehicle- and candesartan-treated animals determined 24 weeks after transplantation. On the other hand, atrophy of tubular cells was significantly attenuated. Candesartan reduced both proteinuria and structural injury of the kidney. In aliskiren-treated animals reduced serum Ang II and Ang (1-7) levels were detected, whereas the level of urine angiotensinogen was unchanged., Conclusions: The renin inhibitor aliskiren does not slow the progression of chronic allograft dysfunction. We suggest that the lack of protection might be due to reduced formation of the protective Ang II breakdown products such as Ang (1-7) or due to unchanged intrarenal RAS activity demonstrated by urinary angiotensinogen levels.

Background: The current study was designed to test the hypothesis that the increased duration of analgesia caused by adding dexmedetomidine to local anesthetic results from blockade of the hyperpolarization-activated cation (I(h)) current., Methods: In this randomized, blinded, controlled study, the analgesic effects of peripheral nerve blocks using 0.5% ropivacaine alone or 0.5% ropivacaine plus dexmedetomidine (34 μM or 6 μg/kg) were assessed with or without the pretreatment of α(1)- and α(2)-adrenoceptor antagonists (prazosin and idazoxan, respectively) and antagonists and agonists of the I(h) current (ZD 7288 and forskolin, respectively). Sciatic nerve blocks were performed, and analgesia was measured by paw withdrawal latency to a thermal stimulus every 30 min for 300 min postblock., Results: The analgesic effect of dexmedetomidine added to ropivacaine was not reversed by either prazosin or idazoxan. There were no additive or attenuated effects from the pretreatment with ZD 7288 (I(h) current blocker) compared with dexmedetomidine added to ropivacaine. When forskolin was administered as a pretreatment to ropivacaine plus dexmedetomidine, there were statistically significant reductions in duration of analgesia at time points 90-180 min (P < 0.0001 for each individual comparison). The duration of blockade for the forskolin (768 μM) followed by ropivacaine plus dexmedetomidine group mirrored the pattern of the ropivacaine alone group, thereby implying a reversal effect., Conclusion: Dexmedetomidine added to ropivacaine caused approximately a 75% increase in the duration of analgesia, which was reversed by pretreatment with an I(h) current enhancer. The analgesic effect of dexmedetomidine was not reversed by an α(2)-adrenoceptor antagonist.

Objective: Gut barrier dysfunction and bacterial translocation occur in various disorders, including intestinal obstruction. Overexpression of inducible nitric oxide synthase is implicated in the pathogenesis of bacterial translocation, of which the molecular mechanism remains unclear. Epithelial permeability is regulated by tight junction reorganization and myosin light chain phosphorylation. Our aim was to investigate the roles of Rho-associated kinase and protein kinase C ζ in epithelial nitric oxide synthase-mediated barrier damage., Design: Animal study and cell cultures., Setting: Research laboratory., Subjects: BALB/c mice., Interventions: : Mouse distal small intestine was obstructed in vivo by a 10-cm loop ligation in which vehicle, L-Nil (a nitric oxide synthase inhibitor), or Y27632 (a Rho-associated kinase inhibitor) was luminally administered. After obstruction for 24 hrs, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 1 mM S-nitroso-N-acetylpenicillamine (a nitric oxide donor) for 24 hrs, and transepithelial resistance and permeability were evaluated., Measurements and Main Results: Mice with intestinal obstruction displayed epithelial barrier dysfunctions, such as permeability rise and bacterial translocation, associated with tight junction disruption and myosin light chain phosphorylation. Increased inducible nitric oxide synthase and phosphorylated protein kinase C ζ were observed in villus epithelium. Enteric instillation of L-Nil and Y27632 attenuated the functional and structural barrier damage caused by intestinal obstruction. L-Nil decreased intestinal obstruction-induced myosin light chain, myosin phosphatase target subunit 1, and protein kinase C ζ phosphorylation, suggesting that inducible nitric oxide synthase is upstream of Rho-associated kinase and protein kinase C ζ signaling. The intestinal phosphorylated myosin light chain level did not increase in inducible nitric oxide synthase(-/-) mice following intestinal obstruction. In vitro studies showed that S-nitroso-N-acetylpenicillamine-induced transepithelial resistance drop and permeability rise was independent of cell apoptosis. Y27632 inhibited S-nitroso-N-acetylpenicillamine-induced myosin light chain phosphorylation and permeability rise. S-nitroso-N-acetylpenicillamine also triggered phosphorylation and membrane translocation of protein kinase C ζ. Inhibitory protein kinase C ζ pseudosubstrate blocked S-nitroso-N-acetylpenicillamine-induced tight junction reorganization, but not myosin light chain phosphorylation., Conclusions: Epithelial inducible nitric oxide synthase activates two distinct signals, protein kinase C ζ and Rho-associated kinase, to disrupt tight junctions leading to bacterial influx.

The search for the mechanism of action of improgan (a nonopioid analgesic) led to the recent discovery of CC12, a compound that blocks improgan antinociception. Because CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (i.c.v.) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) potently inhibited improgan antinociception in rats at doses that were inactive alone. MW06-25, a new P450 inhibitor that combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H(3) receptors, MW06-25 showed no activity at these sites, yet retained potent P450-inhibiting properties. The P450 hypothesis was also tested in Cpr(low) mice, a viable knock-in model with dramatically reduced brain P450 activity. Improgan (145 nmol, i.c.v.) antinociception was reduced by 37% to 59% in Cpr(low) mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, i.c.v.) abolished improgan action (70% to 91%) in control mice, but had no significant effect in Cpr(low) mice. Thus, improgan's activation of bulbospinal nonopioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which (1) improgan activates an unknown receptor to trigger downstream P450 activity, and (2) brainstem epoxygenase activity is a point of convergence for opioid and nonopioid analgesic signaling., (Copyright © 2011 International Association for the Study of Pain. All rights reserved.)

Background: The goal of this in vitro study was to investigate the effects of lipid emulsion (LE) on local anesthetic levobupivacaine-induced responses in isolated rat aorta and to determine whether the effect of LE is related to the lipid solubility of local anesthetics., Methods: Isolated rat aortic rings were suspended for isometric tension recording. The effects of LE were determined during levobupivacaine-, ropivacaine-, and mepivacaine-induced responses. Endothelial nitric oxide synthase and caveolin-1 phosphorylation was measured in human umbilical vein endothelial cells treated with levobupivacaine alone and with the addition of LE., Results: Levobupivacaine produced vasoconstriction at lower, and vasodilation at higher, concentrations, and both were significantly reversed by treatment with LE. Levobupivacaine and ropivacaine inhibited the high potassium chloride-mediated contraction, which was restored by LE. The magnitude of LE-mediated reversal was greater with levobupivacaine treatment than with ropivacaine, whereas this reversal was not observed in mepivacaine-induced responses. In LE-pretreated rings, low-dose levobupivacaine- and ropivacaine-induced contraction was attenuated, whereas low-dose mepivacaine-induced contraction was not significantly altered. Treatment with LE also inhibited the phosphorylation of endothelial nitric oxide synthase induced by levobupivacaine in human umbilical vein endothelial cells., Conclusions: These results indicate that reversal of levobupivacaine-induced vasodilation by LE is mediated mainly through the attenuation of levobupivacaine-mediated inhibition of L-type calcium channel-dependent contraction and, in part, by inhibition of levobupivacaine-induced nitric oxide release. LE-mediated reversal of responses induced by local anesthetics may be related to their lipid solubility.

We investigated the effect of ischemic preconditioning (IPC) on vascular reactivity and calcium sensitivity after hemorrhagic shock and its relationship to the RhoA–Rho-kinase pathway. Using hemorrhagic-shock rats (40 mm Hg for 3 hours) and isolated rings of the superior mesenteric artery (SMA), the effects of IPC (abdominal aorta occlusion applied 2 hours before shock) on the pressor effect of norepinephrine (3 μg/kg), vascular reactivity and calcium sensitivity of SMA, and the activity and role of RhoA and Rho-kinase were investigated. IPC with 1-minute occlusion plus 5-minute loosening of aneurysm clips thrice significantly increased survival time and prevalence of survival at 24 hours of hemorrhagic-shock rats. This IPC condition also significantly increased the pressor response of norepinephrine and significantly improved the vascular reactivity and calcium sensitivity of the SMA. The activity of Rho-kinase and RhoA in the SMA decreased after hemorrhagic shock, but increased after IPC. Y-27632 (Rho-kinase antagonist) and C3 Transferase (RhoA antagonist) significantly decreased IPC-induced increase in vascular reactivity and calcium sensitivity. These results suggested that IPC can improve shock-induced vascular hyporeactivity and calcium desensitization. The RhoA–Rho-kinase pathway played an important part in this process. These findings suggested that the RhoA–Rho-kinase pathway may be a potential target to treat vascular hyporeactivity in severe trauma, shock, or multiple-organ dysfunction syndrome.

As a ubiquitous enzyme overexpressed on the epithelium of the tumor, aminopeptidase N (APN) plays important roles in the angiogenesis and metastasis of the tumor. Bestatin as an effective inhibitor against APN is used in the ancillary treatment of various cancers. In this study, we modified the structure of a bestatin derivative LYP reported in our former study to provide a new bestatin derivative LYP2 with enhanced stability. We also tested the inhibitive activity of LYP2, which retained good efficacy in vitro and in vivo towards APN.