L-arginine and Arginase Products Potentiate Dexmedetomidine-induced Contractions in the Rat Aorta.

BACKGROUND: The α2-adrenergic sedative/anesthetic agent dexmedetomidine exerts biphasic effects on isolated arteries, causing endothelium-dependent relaxations at concentrations at or below 30 nM, followed by contractions at higher concentrations. L-arginine is a common substrate of endothelial nitric oxide synthase and arginases. This study was designed to investigate the role of L-arginine in modulating the overall vascular response to dexmedetomidine. METHODS: Isometric tension was measured in isolated aortic rings of Sprague Dawley rats. Cumulative concentrations of dexmedetomidine (10 nM to 10 µM) were added to quiescent rings (with and without endothelium) after previous incubation with vehicle, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; nitric oxide synthase inhibitor), prazosin (α1-adrenergic antagonist), rauwolscine (α2-adrenergic antagonist), L-arginine, (S)-(2-boronethyl)-L-cysteine hydrochloride (arginase inhibitor), N-hydroxy-L-arginine (arginase inhibitor), urea and/or ornithine. In some preparations, immunofluorescent staining, immunoblotting, or measurement of urea content were performed. RESULTS: Dexmedetomidine did not contract control rings with endothelium but evoked concentration-dependent increases in tension in such rings treated with L-NAME (Emax 50 ± 4%) or after endothelium-removal (Emax 74 ± 5%; N = 7 to 12). Exogenous L-arginine augmented the dexmedetomidine-induced contractions in the presence of L-NAME (Emax 75 ± 3%). This potentiation was abolished by (S)-(2-boronethyl)-L-cysteine hydrochloride (Emax 16 ± 4%) and N-hydroxy-L-arginine (Emax 18 ± 4%). Either urea or ornithine, the downstream arginase products, had a similar potentiating effect as L-arginine. Immunoassay measurements demonstrated an upregulation of arginase I by L-arginine treatment in the presence of L-NAME (N = 4). CONCLUSIONS: These results suggest that when vascular nitric oxide homeostasis is impaired, the potentiation of the vasoconstrictor effect of dexmedetomidine by L-arginine depends on arginase activity and the production of urea and ornithine.

N-acetylcysteine and allopurinol confer synergy in attenuating myocardial ischemia injury via restoring HIF-1α/HO-1 signaling in diabetic rats.

OBJECTIVES: To determine whether or not the antioxidants N-acetylcysteine (NAC) and allopurinol (ALP) confer synergistic cardioprotection against myocardial ischemia/reperfusion (MI/R) injury by stabilizing hypoxia inducible factor 1α (HIF-1α)/heme oxygenase 1 (HO-1) signaling in diabetic myocardium. METHODS: Control or diabetic [streptozotocin (STZ)-induced] Sprague Dawley rats received vehicle or NAC, ALP or their combination for four weeks starting one week after STZ injection. The animals were then subjected to thirty minutes of coronary artery occlusion followed by two hours reperfusion in the absence or presence of the selective HO-1 inhibitor, tin protoporphyrin-IX (SnPP-IX) or the HIF-1α inhibitor 2-Methoxyestradiol (2ME2). Cardiomyocytes exposed to high glucose were subjected to hypoxia/re-oxygenation in the presence or absence of HIF-1α and HO-1 achieved by gene knock-down with related siRNAs. RESULTS: Myocardial and plasma levels of 15-F2t-isoprostane, an index of oxidative stress, were significantly increased in diabetic rats while cardiac HO-1 protein and activity were reduced; this was accompanied with reduced cardiac protein levels of HIF-1α, and increased post-ischemic myocardial infarct size and cellular injury. NAC and ALP given alone and in particular their combination normalized cardiac levels of HO-1 and HIF-1α protein expression and prevented the increase in 15-F2t-isoprostane, resulting in significantly attenuated post-ischemic myocardial infarction. NAC and ALP also attenuated high glucose-induced post-hypoxic cardiomyocyte death in vitro. However, all the above protective effects of NAC and ALP were cancelled either by inhibition of HO-1 or HIF-1α with SnPP-IX and 2ME2 in vivo or by HO-1 or HIF-1α gene knock-down in vitro. CONCLUSION: NAC and ALP confer synergistic cardioprotection in diabetes via restoration of cardiac HIF-1α and HO-1 signaling.

N-Acetylcysteine and allopurinol up-regulated the Jak/STAT3 and PI3K/Akt pathways via adiponectin and attenuated myocardial postischemic injury in diabetes.

N-Acetylcysteine (NAC) and allopurinol (ALP) synergistically reduce myocardial ischemia reperfusion (MI/R) injury in diabetes. However, the mechanism is unclear. We postulated that NAC and ALP attenuated diabetic MI/R injury by up-regulating phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathways subsequent to adiponectin (APN) activation. Control (C) or streptozotocin-induced diabetic rats (D) were untreated or treated with NAC and ALP followed by MI/R. D rats displayed larger infarct size accompanied by decreased phosphorylation of Akt, STAT3 and decreased cardiac nitric oxide (NO) and APN levels. NAC and ALP decreased MI/R injury in D rats, enhanced phosphorylation of Akt and STAT3, and increased NO and APN. High glucose and hypoxia/reoxygenation exposure induced cell death and Akt and STAT3 inactivation in cultured cardiomyocytes, which were prevented by NAC and ALP. The PI3K inhibitor wortmannin and Jak2 inhibitor AG490 abolished the protection of NAC and ALP. Similarly, APN restored posthypoxic Akt and STAT3 activation and decreased cell death in cardiomyocytes. Gene silencing with AdipoR2 siRNA or STAT3 siRNA but not AdipoR1 siRNA abolished the protection of NAC and ALP. In conclusion, NAC and ALP prevented diabetic MI/R injury through PI3K/Akt and Jak2/STAT3 and cardiac APN may serve as a mediator via AdipoR2 in this process.

Hyperglycemia-induced protein kinase C ß2 activation induces diastolic cardiac dysfunction in diabetic rats by impairing caveolin-3 expression and Akt/eNOS signaling.

Protein kinase C (PKC)ß2 is preferably overexpressed in the diabetic myocardium, which induces cardiomyocyte hypertrophy and contributes to diabetic cardiomyopathy, but the underlying mechanisms are incompletely understood. Caveolae are critical in signal transduction of PKC isoforms in cardiomyocytes. Caveolin (Cav)-3, the cardiomyocyte-specific caveolar structural protein isoform, is decreased in the diabetic heart. The current study determined whether PKCß2 activation affects caveolae and Cav-3 expression. Immunoprecipitation and immunofluorescence analysis revealed that high glucose (HG) increased the association and colocalization of PKCß2 and Cav-3 in isolated cardiomyocytes. Disruption of caveolae by methyl-ß-cyclodextrin or Cav-3 small interfering (si)RNA transfection prevented HG-induced PKCß2 phosphorylation. Inhibition of PKCß2 activation by compound CGP53353 or knockdown of PKCß2 expression via siRNA attenuated the reductions of Cav-3 expression and Akt/endothelial nitric oxide synthase (eNOS) phosphorylation in cardiomyocytes exposed to HG. LY333531 treatment (for a duration of 4 weeks) prevented excessive PKCß2 activation and attenuated cardiac diastolic dysfunction in rats with streptozotocin-induced diabetes. LY333531 suppressed the decreased expression of myocardial NO, Cav-3, phosphorylated (p)-Akt, and p-eNOS and also mitigated the augmentation of O2(-), nitrotyrosine, Cav-1, and iNOS expression. In conclusion, hyperglycemia-induced PKCß2 activation requires caveolae and is associated with reduced Cav-3 expression in the diabetic heart. Prevention of excessive PKCß2 activation attenuated cardiac diastolic dysfunction by restoring Cav-3 expression and subsequently rescuing Akt/eNOS/NO signaling.

N-acetylcysteine and allopurinol synergistically enhance cardiac adiponectin content and reduce myocardial reperfusion injury in diabetic rats.

BACKGROUND: Hyperglycemia-induced oxidative stress plays a central role in the development of diabetic myocardial complications. Adiponectin (APN), an adipokine with anti-diabetic and anti-ischemic effects, is decreased in diabetes. It is unknown whether or not antioxidant treatment with N-acetylcysteine (NAC) and/or allopurinol (ALP) can attenuate APN deficiency and myocardial ischemia reperfusion (MI/R) injury in the early stage of diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Control or streptozotocin (STZ)-induced diabetic rats were either untreated (C, D) or treated with NAC (1.5 g/kg/day) or ALP (100 mg/kg/day) or their combination for four weeks starting one week after STZ injection. Plasma and cardiac biochemical parameters were measured after the completion of treatment, and the rats were subjected to MI/R by occluding the left anterior descending artery for 30 min followed by 2 h reperfusion. Plasma and cardiac APN levels were decreased in diabetic rats accompanied by decreased cardiac APN receptor 2 (AdipoR2), reduced phosphorylation of Akt, signal transducer and activator of transcription 3 (STAT3) and endothelial nitric oxide synthase (eNOS) but increased IL-6 and TNF-α (all P<0.05 vs. C). NAC but not ALP increased cardiac APN concentrations and AdipoR2 expression in diabetic rats. ALP enhanced the effects of NAC in restoring cardiac AdipoR2 and phosphorylation of Akt, STAT3 and eNOS in diabetic rats. Further, NAC and ALP, respectively, decreased postischemic myocardial infarct size and creatinine kinase-MB (CK-MB) release in diabetic rats, while their combination conferred synergistic protective effects. In addition, exposure of cultured rat cardiomyocytes to high glucose resulted in significant reduction of cardiomyocyte APN concentration and AdipoR2 protein expression. APN supplementation restored high glucose induced AdipoR2 reduction in cardiomyocytes. CONCLUSIONS/SIGNIFICANCE: NAC and ALP synergistically restore myocardial APN and AdipoR2 mediated eNOS activation. This may represent the mechanism through which NAC and ALP combination greatly reduces MI/R injury in early diabetic rats.

Reducing perioperative blood loss and allogeneic blood transfusion in patients undergoing major spine surgery.

At present, individual techniques, including intraoperative acute normovolemic hemodilution, use of tranexamic acid, use of intrathecal morphine, proper positioning, and modification of operative techniques, seem most promising for reducing perioperative blood loss and allogeneic blood transfusion in patients undergoing major spine surgery. Other techniques including preoperative autologous predonation; mandatory discontinuation of use of antiplatelet agents; intraoperative and postoperative red-blood-cell salvage; use of aprotinin, epsilon-aminocaproic acid, recombinant factor VIIa, or desmopressin; induced hypotension; avoidance of hypothermia; and minimally invasive operative techniques require additional studies to either establish their effectiveness or address safety considerations.

Dexmedetomidine induces both relaxations and contractions, via different {alpha}2-adrenoceptor subtypes, in the isolated mesenteric artery and aorta of the rat.

Dexmedetomidine is an α(2)-adrenoceptor agonist and anesthetic. The present study was designed to characterize the receptor subtypes and the downstream mechanisms of the vascular effects of dexmedetomidine in small (mesenteric artery) and large (aorta) arteries ex vivo. Isometric tension was measured in Sprague-Dawley rat mesenteric and aortic rings (with or without endothelium). To study relaxations, cumulative concentrations of dexmedetomidine, 5-bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine, (UK14304), or clonidine were added to rings contracted with 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F(2α) (U46619) in the presence or absence of indomethacin; N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME); 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate (BRL44408); 2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239); l-657,743, (2S-trans)-1,3,4,5',6,6',7,12b-octahydro-1',3'-dimethyl-spiro[2H-benzofuro[2,3-a]quinolizine-2,4'(1'H)-pyrimidin]-2'(3'H)-one hydrochloride hydrate (MK912); rauwolscine; prazosin; or pertussis toxin. To study contractions, dexmedetomidine was added to quiescent rings without endothelium or after incubation with l-NAME, rauwolscine, prazosin, indomethacin, or 3-[(6-amino-(4-chlorobenzensulfonyl)-2-methyl-5,6,7,8-tetrahydronaphth)-1-yl]propionic acid (S18886). Dexmedetomidine evoked relaxation at low concentrations (10 pM-30 nM) followed by contraction at higher concentrations (>30 nM) in the mesenteric artery. In the aorta, the relaxation was significantly smaller. The relaxation to dexmedetomidine depended on nitric oxide, endothelium, and G(i) protein, and it was mediated by α(2A/D)-adrenoceptors and possibly α(2B)-adrenoceptors. The contraction was mediated mainly by α(2B)- and α(1)-adrenoceptors and involved the action of prostanoids. UK14304 and clonidine induced greater and smaller relaxations, respectively, than dexmedetomidine. In conclusion, depending on the concentration used and the presence of functional endothelium, dexmedetomidine may evoke both relaxation and contraction in isolated arteries. The vascular effects also vary depending on the blood vessel studied. Its vascular effect is different from that of clonidine and UK14304.

Comprehensive preoperative evaluation of platelet function in total knee arthroplasty patients taking diclofenac.

The severity and variability of platelet dysfunction in preoperative arthritic patients taking nonsteroidal anti-inflammatory drugs (NSAIDs) have not been well studied previously. We evaluate 30 preoperative patients taking diclofenac (group D) by routine coagulation screen, platelet count, fibrinogen concentration, thrombelastography, and PFA-100 (Dade Behring, Inc, Deerfield, IL)) platelet function analyzer. Ten patients (group P) and 30 healthy volunteers (group N) not taking NSAIDs serve as control. Diclofenac causes significant prolongation of mean PFA-100 closure times (P < .0001). However, the prolongation is highly variable; and up to 33% of patients are still having normal platelet function despite diclofenac consumption. Low body weight is a significant predictor of more severe platelet dysfunction (P < .01). Other tests are not useful. We conclude that not all patients taking NSAIDs have similar platelet dysfunction and that preoperative monitoring with PFA-100 is preferable.

Genistein potentiates protein kinase A activity in porcine coronary artery.

Soy consumption is associated with a lower risk of atherosclerotic disease in the oriental population. Genistein is a soy isoflavone bearing estrogenic properties. Previous experiments in our laboratory demonstrated the potentiation of endothelium-independent relaxation of coronary artery by both estrogen and genistein. The potentiating effects of both estrogen and genistein were mediated through the cAMP-signaling pathway. We hypothesize that genistein could enhance protein kinase A (PKA) activity in porcine coronary artery smooth muscle, thereby offering a mechanism for the potentiation of vascular relaxation by genistein. In our study, a high concentration of genistein (10(-4.5) M) significantly increased PKA activity in porcine coronary artery rings. While genistein at 10(-5.5) M and forskolin at 10(-7) M had no effect on PKA activity, the combination of the two compounds at the prescribed concentrations caused a significant increase in PKA activity. The increase in PKA activity by genistein was abolished by SQ 22536 (adenylate cyclase blocker), but not by NF 449 (Gs protein blocker) or ICI 182780 (estrogen receptor antagonist). Our results suggest that the action of genistein is mediated via adenylate cyclase, but does not appear to involve Gs protein or ICI 182780-sensitive estrogen receptor.

The potential use of desmopressin to correct hypothermia-induced impairment of primary haemostasis--an in vitro study using PFA-100.

OBJECTIVE: Mild hypothermia (32-35 degrees C) impairs primary haemostasis and coagulation. Correction of these haemostatic impairments by rewarming alone may not be possible or desirable, particularly in major trauma, neuroanaesthesia and in critically ill patients. Pharmacological treatment of these impairments, if available, may be a useful alternative. Desmopressin has been used to treat various congenital and acquired platelet disorders, but its effects on hypothermia-induced impairment of primary haemostasis is not known. This study aims to investigate the in vitro effects of desmopressin on hypothermia-induced impairment of primary haemostasis using PFA-100 platelet function analyzer. METHODS: Whole blood was collected from 20 healthy volunteers, divided into 2.7 ml aliquots and some incubated at 32 degrees C, and others at 37 degrees C as control. Three log doses of desmopressin (0.01, 0.1 or 1 nM) were added to aliquots at 32 degrees C, and saline was added to controls at both 32 and 37 degrees C, all in 0.1 ml volume. After incubating for 30 min, closure times (CT) was measured by PFA-100 using both collagen/epinephrine (adrenaline) (Col/EPI) and collagen/adenosine-5'-diphosphate (Col/ADP) cartridges. RESULTS: CT was prolonged by 30.9% (Col/EPI) and 18.8% (Col/ADP) at 32 degrees C, respectively, compared to 37 degrees C (P<0.001). All the three doses of desmopressin significantly, but incompletely corrected CT prolongation due to hypothermia (P<0.002). CONCLUSION: Desmopressin partially reverses hypothermia-induced impairment of primary haemostasis in vitro, and may be potentially useful in improving haemostasis in hypothermic patients with bleeding where immediate rewarming is difficult or undesirable.

A comparison of postoperative cognitive function and pain relief with fentanyl or tramadol patient-controlled analgesia.

STUDY OBJECTIVE: The use of different opioids for patient-controlled analgesia (PCA) may affect postoperative cognitive function differently. Patient-controlled analgesia fentanyl has been shown to preserve cognitive function better than morphine. The effect of PCA tramadol on cognitive function is unknown. This study aims to compare postoperative cognitive function and analgesia of PCA fentanyl or tramadol. DESIGN: Prospective randomized double-blinded study. SETTING: Metropolitan teaching hospital. PATIENTS: 30 ASA physical status I, II, and III patients undergoing lower abdominal operations. INTERVENTIONS: Patients received standard general anesthesia for their operations. Postoperatively, patients received either fentanyl (group F, 10 mug bolus, n = 17) or tramadol (group T, 20 mg bolus, n = 13) for PCA. Group F patients also received fentanyl boluses and group T patients received tramadol boluses intraoperatively. MEASUREMENTS: Cognitive function was measured using Mini-Mental State Examination and Benton Visual Retention Test (BVRT) preoperatively and on days 1 and 2. Pain was measured by numerical rating scale. RESULTS: No differences were found in postoperative Mini-Mental State Examination or BVRT scores, but significantly fewer (29.4%; 95% confidence interval [CI], 13.3%-53.1%) group F patients were able to complete BVRT compared with group T patients (84.6%; 95% CI, 57.8%-95.7%; 95% CI of difference, 19.4%-74.8%) (P = 0.010) on day 1. In the first 24 hours, group F and group T patients had similar analgesia at rest, but group T patients had better analgesia during cough (mean Numeric Rating Scale, 7.6; 95% CI, 7.0-8.2 vs 6.0; 95% CI, 4.8-7.2, group F vs group T) (P = 0.018; 95% CI of difference, 0.4-2.8). No differences were found in frequency of side effects or patient satisfaction. CONCLUSIONS: Tramadol or fentanyl PCA has similar cognitive effects on days 1 and 2; however, patients receiving tramadol PCA are more motivated to undergo cognitively demanding tasks and have slightly better analgesia on postoperative day 1.

A prospective, randomized, double-blind, placebo-controlled study of the platelet and global hemostatic effects of Ganoderma lucidum (Ling-Zhi) in healthy volunteers.

UNLABELLED: Ganoderma lucidum is a Chinese herbal medicine popular with cancer patients. Previous in vitro studies suggested that Ganoderma lucidum might impair hemostasis. In this prospective, randomized double-blind study, healthy volunteers received orally Ganoderma lucidum capsules 1.5 g (n = 20) or placebo (n = 20) daily for 4 wk. We monitored subjects before drug administration and at 4 and 8 wk thereafter by routine coagulation screen, fibrinogen concentration, von Willebrand ristocetin cofactor activity, platelet function analyzer PFA-100, and thrombelastography. There were no significant between-group differences and all measurements remained within the normal range. Ganoderma lucidum ingestion over 4 wk was not associated with impairment of hemostasis. IMPLICATIONS: Ingestion of Ganoderma lucidum does not cause impairment of hemostatic function in healthy volunteers, despite earlier in vitro reports that it may cause platelet inhibition and may have other antithrombotic and fibrinolytic activity. The use of Ganoderma lucidum preoperatively is unlikely to increase the risk of surgical bleeding in otherwise healthy patients.

Changes in thrombelastograph variables associated with aging.

Aging is associated with hypercoagulability. To assess thrombelastography (TEG) variables associated with aging, 132 adult patients of various ages undergoing orthopedic surgery for fracture repair had venous blood samples withdrawn for testing of recalcified TEG before the induction of anesthesia. Age was weakly correlated with all TEG variables: r time (R) (r = -0.45, P < 0.001; R = 19.5 - 0.09 x age), k time (K) (r = -0.49, P < 0.001; K = 6.5 - 0.04 x age), maximum amplitude (MA) (r = 0.25, P < 0.01; MA = 53.3 + 0.07 x age), and alpha (r = 0.52, P < 0.001; alpha = 52.8 + 0.2 x age). The correlation was stronger for men than for women. Only R was significantly correlated with age when the women were separately analyzed. Part of the correlation may be attributable to a concurrent decrease in hemoglobin with aging, but age remained an independent predictor of R, K, and alpha on forward stepwise linear multiple regression analysis. Aging was weakly associated with changes in TEG variables, which should be allowed for when interpreting TEG measurements in the elderly.

Value of preoperative coagulation tests: reappraisal of major noncardiac surgery.

In a retrospective case-control review, we evaluated preoperative coagulation testing in patients undergoing major noncardiac operations to determine if routine testing benefits this group of patients. The platelet count (PC), prothrombin time (PT), and activated partial thromboplastin time (aPTT) in all patients undergoing major noncardiac surgery over a 22-month period were reviewed. The review was done both manually and by the computerized hospital information system. Major surgery was defined as procedures usually associated with significant bleeding. For each patient with abnormal results, another two control patients undergoing the same surgery and matched for age and gender were identified. Case and control patients were compared regarding a change in the management plan, use of blood products, blood loss, and bleeding complications by detailed chart review. A total of 828 patients undergoing nine different surgeries were reviewed. The incidence of abnormal PCs was 2.2% [95% confidence interval (CI) 1.2-3.2%] and that of abnormal PT/aPTTs was 2.1% (95% CI 1.1-3.1%). There were only two cases each of thrombocytopenia and prolonged PT/aPTT where the coagulation tests were not indicated clinically. Although (compared to controls) patients with abnormal tests had more changes in their anesthesia plan (36% vs. 2%, p < 0.001) and platelet or fresh frozen plasma transfusions (50% vs. 9%, p < 0.001), blood loss and the incidence of bleeding complications were not different. We conclude that the use of preoperative coagulation tests in patients undergoing major noncardiac surgery should still be guided by clinical assessment. The surgical procedure itself does not constitute an indication for testing.