Curcumin Stereoisomer, Cis-Trans Curcumin, as a Novel Ligand to A1 and A3 Adenosine Receptors.

Adenosine receptors (ARs) are being explored to generate non-opioid pain therapeutics. Vanilloid compounds, curcumin, capsaicin, and vanillin possess antinociceptive properties through their interactions with the transient receptor potential channel family. However, their binding with adenosine receptors has not been well studied. The hypothesis in this study was that a vanilloid compound, cis-trans curcumin (CTCUR), binds to each of the two Gi-linked AR subtypes (A1AR and A3AR). CTCUR was synthesized from curcumin (CUR) using the cavitand-mediated photoisomerization technique. The cell lines transfected with the specific receptor (A1AR or A3AR) were treated with CTCUR or CUR and the binding was analyzed using competitive assays, confocal microscopy, and docking. The binding assays and molecular docking indicated that CTCUR had Ki values of 306 nM (A1AR) and 400 nM (A3AR). These values suggest that CTCUR is selective for Gi-linked ARs (A1AR or A3AR) over Gs-linked ARs (A2AAR or A2BAR), based on our previous published research. In addition, the docking showed that CTCUR binds to the toggle switch domain of ARs. Curcumin (CUR) did not exhibit binding at any of these receptors. In summary, CTCUR and other modifications of CUR can be developed as novel therapeutic ligands for the Gi-linked ARs (A1AR and A3AR) involved with pain and cancer.

Synthesis of ribavirin 1,2,3- and 1,2,4-triazolyl analogs with changes at the amide and cytotoxicity in breast cancer cell lines.

We report the synthesis and cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells of novel 1,2,3- and 1,2,4-triazolyl analogs of ribavirin. We modified ribavirin's carboxamide moiety to test the effects of lipophilic groups. 1-ß-D-Ribofuranosyl-1H-1,2,3-triazoles were prepared using Click Chemistry, whereas an unprecedented application of a prior 1,2,4-triazole ring synthesis was used for 1-ß-D-ribofuranosyl-1H-1,2,4-triazole analogs. Though cytotoxicity was mediocre and there was no correlation with lipophilicity, we discovered that a structurally similar concentrative nucleoside transporter 2 (CNT2) inhibitor was modestly cytotoxic (MCF-7 IC50 of 42 µM). These syntheses could be used to efficiently investigate variation in the nucleobase.

Spirocyclic dimer SpiD7 activates the unfolded protein response to selectively inhibit growth and induce apoptosis of cancer cells.

The unfolded protein response (UPR) is an adaptation mechanism activated to resolve transient accumulation of unfolded/misfolded proteins in the endoplasmic reticulum. Failure to resolve the transient accumulation of such proteins results in UPR-mediated programmed cell death. Loss of tumor suppressor gene or oncogene addiction in cancer cells can result in sustained higher basal UPR levels; however, it is not clear if these higher basal UPR levels in cancer cells can be exploited as a therapeutic strategy. We hypothesized that covalent modification of surface-exposed cysteine (SEC) residues could simulate unfolded/misfolded proteins to activate the UPR, and that higher basal UPR levels in cancer cells would provide the necessary therapeutic window. To test this hypothesis, here we synthesized analogs that can covalently modify multiple SEC residues and evaluated them as UPR activators. We identified a spirocyclic dimer, SpiD7, and evaluated its effects on UPR activation signals, that is, XBP1 splicing, phosphorylation of eIF2α, and a decrease in ATF 6 levels, in normal and cancer cells, which were further confirmed by RNA-Seq analyses. We found that SpiD7 selectively induced caspase-mediated apoptosis in cancer cells, whereas normal cells exhibited robust XBP1 splicing, indicating adaptation to stress. Furthermore, SpiD7 inhibited the growth of high-grade serous carcinoma cell lines ~3-15-fold more potently than immortalized fallopian tube epithelial (paired normal control) cells and reduced clonogenic growth of high-grade serous carcinoma cell lines. Our results suggest that induction of the UPR by covalent modification of SEC residues represents a cancer cell vulnerability and can be exploited to discover novel therapeutics.

Regulating Polyamine Metabolism by miRNAs in Diabetic Cardiomyopathy.

PURPOSE OF REVIEW: Insulin is at the heart of diabetes mellitus (DM). DM alters cardiac metabolism causing cardiomyopathy, ultimately leading to heart failure. Polyamines, organic compounds synthesized by cardiomyocytes, have an insulin-like activity and effect on glucose metabolism, making them metabolites of interest in the DM heart. This review sheds light on the disrupted microRNA network in the DM heart in relation to developing novel therapeutics targeting polyamine biosynthesis to prevent/mitigate diabetic cardiomyopathy. RECENT FINDINGS: Polyamines prevent DM-induced upregulation of glucose and ketone body levels similar to insulin. Polyamines also enhance mitochondrial respiration and thereby regulate all major metabolic pathways. Non-coding microRNAs regulate a majority of the biological pathways in our body by modulating gene expression via mRNA degradation or translational repression. However, the role of miRNA in polyamine biosynthesis in the DM heart remains unclear. This review discusses the regulation of polyamine synthesis and metabolism, and its impact on cardiac metabolism and circulating levels of glucose, insulin, and ketone bodies. We provide insights on potential roles of polyamines in diabetic cardiomyopathy and putative miRNAs that could regulate polyamine biosynthesis in the DM heart. Future studies will unravel the regulatory roles these miRNAs play in polyamine biosynthesis and will open new doors in the prevention/treatment of adverse cardiac remodeling in diabetic cardiomyopathy.

Hyperglycemic conditions proliferate triple negative breast cancer cells: role of ornithine decarboxylase.

PURPOSE: Several cancer subtypes (pancreatic, breast, liver, and colorectal) rapidly advance to higher aggressive stages in diabetes. Though hyperglycemia has been considered as a fuel for growth of cancer cells, pathways leading to this condition are still under investigation. Cellular polyamines can modulate normal and cancer cell growth, and inhibitors of polyamine synthesis have been approved for treating colon cancer, however the role of polyamines in diabetes-mediated cancer advancement is unclear as yet. We hypothesized that polyamine metabolic pathway is involved with increased proliferation of breast cancer cells under high glucose (HG) conditions. METHODS: Studies were performed with varying concentrations of glucose (5-25 mM) exposure in invasive, triple negative breast cancer cells, MDA-MB-231; non-invasive, estrogen/progesterone receptor positive breast cancer cells, MCF-7; and non-tumorigenic mammary epithelial cells, MCF-10A. RESULTS: There was a significant increase in proliferation with HG (25 mM) at 48-72 h in both MDA-MB-231 and MCF-10A cells but no such effect was observed in MCF-7 cells. This was correlated to higher activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine synthesis pathway. Inhibitor of polyamine synthesis (difluoromethylornithine, DFMO, 5 mM) was quite effective in suppressing HG-mediated cell proliferation and ODC activity in MDA-MB-231 and MCF-10A cells. Polyamine (putrescine) levels were significantly elevated with HG treatment in MDA-MB-231 cells. HG exposure also increased the metastasis of MDA-MB-231 cells. CONCLUSIONS: Our cellular findings indicate that polyamine inhibition should be explored in patient population as a target for future chemotherapeutics in diabetic breast cancer.

Novel curcumin analog (cis-trans curcumin) as ligand to adenosine receptors A2A and A2B: potential for therapeutics.

All four of the adenosine receptor (AR) subtypes mediate pain and have been targeted by pharmacologists to generate new therapeutics for chronic pain. The vanilloid phytochemicals, which include curcumin, capsaicin, and gingerol, have been shown to alleviate pain. However, there is little to no literature on the interaction of vanilloid phytochemicals with ARs. In this study, photochemical methods were used to generate a novel isomer of curcumin (cis-trans curcumin or CTCUR), and the interactions of both curcumin and CTCUR with the two Gs-linked AR subtypes were studied. Competitive binding assays, docking analysis, and confocal fluorescence microscopy were performed to measure binding affinity; cell survival assays were used to measure toxicity; and cAMP assays were performed to measure receptor activation. Competitive binding results indicated that CTCUR binds to both AR A2A and AR A2B with Ki values of 5 µM and 7 µM, respectively, which is consistent with our docking results. Fluorescence microscopy data also shows binding for A2B and A2A. Cell survival results show that CTCUR and CUR are nontoxic at the tested concentrations in these cell lines. Overall, our results suggest that vanilloid phytochemicals may be slightly modified to increase interaction with Gs-ARs, and thereby can be further explored to provide a novel class of non-opioid antinociceptives.

The Effects of Prodrug Size and a Carbonyl Linker on l-Type Amino Acid Transporter 1-Targeted Cellular and Brain Uptake.

The l-type amino acid transporter 1 (LAT1, SLC7A5) imports dietary amino acids and amino acid drugs (e. g., l-DOPA) into the brain, and plays a role in cancer metabolism. Though there have been numerous reports of LAT1-targeted amino acid-drug conjugates (prodrugs), identifying the structural determinants to enhance substrate activity has been challenging. In this work, we investigated the position and orientation of a carbonyl group in linking hydrophobic moieties including the anti-inflammatory drug ketoprofen to l-tyrosine and l-phenylalanine. We found that esters of meta-carboxyl l-phenylalanine had better LAT1 transport rates than the corresponding acylated l-tyrosine analogues. However, as the size of the hydrophobic moiety increased, we observed a decrease in LAT1 transport rate with a concomitant increase in potency of inhibition. Our results have important implications for designing amino acid prodrugs that target LAT1 at the blood-brain barrier or on cancer cells.

Biomechanical and Biophysical Properties of Breast Cancer Cells Under Varying Glycemic Regimens.

Diabetes accelerates cancer cell proliferation and metastasis, particularly for cancers of the pancreas, liver, breast, colon, and skin. While pathways linking the 2 disease conditions have been explored extensively, there is a lack of information on whether there could be cytoarchitectural changes induced by glucose which predispose cancer cells to aggressive phenotypes. It was thus hypothesized that exposure to diabetes/high glucose alters the biomechanical and biophysical properties of cancer cells more than the normal cells, which aids in advancing the cancer. For this study, atomic force microscopy indentation was used through microscale probing of multiple human breast cancer cells (MCF-7, MDA-MB-231), and human normal mammary epithelial cells (MCF-10A), under different levels of glycemic stress. These were used to study both benign and malignant breast tissue behaviors. Benign cells (MCF-10A) recorded higher Young's modulus values than malignant cells (MCF-7 and MDA-231) under normoglycemic conditions, which agrees with the current literature. Moreover, exposure to high glucose (for 48 hours) decreased Young's modulus in both benign and malignant cells, to the effect that the cancer cells showed a complete loss in elasticity with high glucose. This provides a possible insight into a link between glycemic stress and cytoskeletal strength. This work suggests that reducing glycemic stress in cancer patients and those at risk can prove beneficial in restoring normal cytoskeletal structure.

Early Neuromuscular Blockade in Children with Pediatric Acute Respiratory Distress Syndrome.

Pediatric acute respiratory distress syndrome (PARDS) is a challenging problem with high mortality. Role of neuromuscular blockade in the management of ARDS to date has been controversial, and this study was done to study the role of neuromuscular blockade in children having PARDS and development of associated complications, if any. This was a prospective, case-control study conducted in the pediatric intensive care unit (PICU) of a tertiary care teaching hospital, over a period of 24 months. Patients of age 1 to 18 years who presented with or developed PARDS during their course of hospitalization were included after written informed consent was obtained from their parents and/or guardians. Patients with PARDS requiring invasive mechanical ventilation were partitioned into a case group and a control group. Case group patients were sedated and paralyzed using midazolam (1 µg/kg/min) and vecuronium (1 µg/kg/min), respectively, along with institution of definitive management. Control group patients were given definitive and supportive therapy, but no neuromuscular blocking agents (NMBAs). All patients were followed up for signs and symptoms of myopathy or neuropathy during the entire duration of hospital stay and up to 3 months after discharge. During the study period, 613 patients were admitted to the PICU of which 91 patients qualified as having PARDS. Sepsis was the main etiology in 67 of the 91 patients (73.6%) with PARDS. Fifty-nine patients were included in the study, of which 29 patients were included in the case group and 30 patients were included in the control group. Among the 29 case group patients, 25 patients (86.2%) were successfully extubated. Four patients from the case group expired, while 14 out of 30 control group patients (46.7%) expired. Hypotension was present in 26 case group patients (89.6%), of which all showed resolution within 48 hours of definitive treatment. The mean time to resolution of hypotension was 41.6 hours (standard deviation [SD]: 5.759; range: 24-48) for case group patients, significantly lower ( p < 0.0001) than the mean time to resolution of 103 hours (SD: 18.995; range: 90-126) for the 10 control group patients with hypotension that survived. Mean oxygenation index (OI) following 48 hours of vecuronium therapy was significantly lower ( p < 0.0001; 95% confidence interval: 5.9129-9.9671) than mean OI at admission for case group patients. None of the patients receiving vecuronium exhibited neuromuscular deficit during their hospital stay, at time of discharge, or at follow-up evaluation up to 3 months after discharge. In this study, pediatric cases diagnosed with PARDS and managed with mechanical ventilation and vecuronium therapy had improved mean OI following 48 hours of NMBA therapy and a lower mortality when compared with matched control group patients. Incidence of NMBA-related weakness was not commonly observed in these patients.

Hyperglycemia-impaired aortic vasorelaxation mediated through arginase elevation: Role of stress kinase pathways.

Diabetes-induced vascular endothelial dysfunction has been reported to involve hyperglycemia-induced increases in arginase activity. However, upstream mediators of this effect are not clear. Here, we have tested involvement of Rho kinase, ERK1/2 and p38 MAPK pathways in this process. Studies were performed with aortas isolated from wild type or hemizygous arginase 1 knockout (Arg1+/-) mice and bovine aortic endothelial cells exposed to high glucose (HG, 25 mmol/l) or normal glucose (NG, 5.5 mmol/l) conditions for different times. Effects of inhibitors of arginase, p38 MAPK, ERK1/2 or ROCK and ex vivo adenoviral delivery of active Arg1 and inactive (D128-Arg1) cDNA were also determined. Exposure in wild type aorta or endothelial cells to HG significantly increased arginase activity and Arg1 expression and impaired aortic relaxation. Transduction of wild type aorta with active Arg1 cDNA impaired vascular relaxation, whereas inactive Arg1 had no effect. The HG-induced vascular endothelial dysfunction was associated with increased phosphorylation (activation) of ERK1/2 and p38 MAPK. Pretreatment with inhibitors of ERK1/2, p38 MAPK, ROCK or arginase blocked HG-induced elevation of arginase activity and Arg1 expression and prevented the vascular dysfunction. Inhibition of ROCK blunted the HG-induced activation of ERK1/2 and p38 MAPK. In summary, activated ROCK and subsequent activation of ERK1/2 or p38 MAPK elevates arginase activity and Arg1 expression in hyperglycemic states. Targeting this pathway may provide an effective means for preventing diabetes/hyperglycemia-induced vascular endothelial dysfunction.

Differential Mobility of Breast Cancer Cells and Normal Breast Epithelial Cells Under DC Electrophoresis and Electroosmosis.

BACKGROUND/AIM: Breast cancer has shown improved prognosis over the years, yet due to different types of breast cancer and different phenotypes of breast cancer cells, it is essential to have an accurate diagnostic procedure. MATERIALS AND METHODS: A unique methodology employing direct current (DC) electrophoresis and electroosmosis in fabricated microfluidic devices was used. This phenomenon was used to discern breast cancer cells from normal breast epithelial cells based on their electrophoretic mobility and electroosmosis. RESULTS: Breast cancer cells (MDA-MB-231) rapidly covered the wound in scratch assay compared to mammary epithelial cells (MCF-10A). However, under DC electrophoresis and electroosmosis MCF-10A cells were observed to move at higher velocities compared to MDA-MB-231 cells under similar voltages. CONCLUSION: Our study provides an economical and efficient method of studying electrophoretic and electrokinetic phenomenon of different cell types that can be used to the advantage of detecting and monitoring cancer progression.

Antineoplastic Actions of Cinnamic Acids and Their Dimers in Breast Cancer Cells: A Comparative Study.

BACKGROUND/AIM: Breast cancer is the second leading cause of cancer-related deaths in US, which necessitates constant research for medications with minimal adverse effects. The aim of the study was to determine if certain cinnamic acid dimers (CADs) exhibit higher cytotoxicity in breast cancer cells than against non-tumorigenic cells. MATERIALS AND METHODS: These dimers have not been explored, so far, due to their complex stereochemistry; cavitand-mediated photodimerization (CMP) method was used to produce several CADs, which were tested for cytotoxicity, apoptosis and cell cycle inhibition. RESULTS: CADs such as ferulic acid dimer, 3-fluoro CAD, and 3, 4-difluoro CAD, were found to be more cytotoxic than their parent monomers. The most potent compound, 3-fluoro CAD caused apoptosis and 'S' phase cell cycle arrest in cancer cells. CONCLUSION: This study indicates an avenue for research for developing a novel class of antineoplastic compounds that can be synthesized using an easy, economical method.

Ferulic acid dimer as a non-opioid therapeutic for acute pain.

PURPOSE: Search for alternate pain medications has gained more importance in the past few years due to adverse effects associated with currently prescribed drugs including nervous system dysfunction with opioids, gastrointestinal discomfort with nonsteroidal anti-inflammatory drugs, and cardiovascular anomalies with cyclooxygenase-2 (COX-2) inhibitors. Phytomedicine has been explored for the treatment of pain, as these have been used for generations in regional communities and tend to lack major side effects in general. One such phytomedicine, incarvillateine (INCA), derived from the Chinese herb Incarvillea sinensis has its primary antinociceptive action through the adenosine receptor, a novel pain target. We hypothesized that derivatives of cinnamic acid dimers, which are structurally similar to INCA, would show potent antinociceptive action and that their effect would be mediated through adenosine receptor action. MATERIALS AND METHODS: Dimers of cinnamic acid (INCA analogs) were synthesized using cavitand-mediated photodimerization (CMP) method, which utilizes a macromolecule (γ-cyclodextrin) to control excited state reactivity of photoactive compounds. Acute pain response was assessed by using formalin-induced licking behavior in hind paw of mice, and neurologic function was monitored through locomotor activity, mechanical hyperalgesia, and thermal sensitivity upon administration of test compound. For mechanistic studies, binding to adenosine receptor was determined by using computer modeling. RESULTS: Ferulic acid dimer (FAD), which has the same chemical functionalities on the aromatic ring as INCA, showed significant suppression of formalin-induced acute pain. Antinociceptive effect was observed primarily in the inflammatory phase, and no apparent behavioral changes related to the nervous system were noticeable. Inhibition of opioid receptor did not reverse antinociceptive response, and modeling data suggest adenosine 3 receptor binding. CONCLUSION: FAD (INCA analog) shows potent nonopioid antinociceptive action mediated predominantly through A3AR - adenosine 3 receptor action. Further characterization and selection of such INCA analogs will help us generate a new class of antinociceptives with precise chemical modifications by using CMP methodology.

Prevention of diabetes-induced arginase activation and vascular dysfunction by Rho kinase (ROCK) knockout.

AIMS: We determined the role of the Rho kinase (ROCK) isoforms in diabetes-induced vascular endothelial dysfunction and enhancement of arginase activity and expression. METHODS AND RESULTS: Studies were performed in aortic tissues from haplo-insufficient (H-I) ROCK1 and ROCK2 mice and wild-type (WT) mice rendered diabetic with streptozotocin and in bovine aortic endothelial cells (BAECs) treated with high glucose (HG, 25 mM). Protein expression of both ROCK isoforms was substantially elevated in aortas of WT mice after 8 weeks of diabetes and in BAECs after 48 h in HG. Impairment of endothelium-dependent vasorelaxation of aortas was observed in diabetic WT mice. However, there was no impairment in aortas of diabetic ROCK1 H-I mice and less impairment in aortas of diabetic ROCK2 H-I mice, compared with non-diabetic mice. These vascular effects were associated with the prevention of diabetes-induced decrease in nitric oxide (NO) production and a rise in arginase activity/expression. Acute treatment with the arginase inhibitor, BEC, improved endothelium-dependent vasorelaxation of aortas of both diabetic WT and ROCK2, but not of ROCK1 mice. CONCLUSION: Partial deletion of either ROCK isoform, but to a greater extent ROCK1, attenuates diabetes-induced vascular endothelial dysfunction by preventing increased arginase activity and expression and reduction in NO production in type 1 diabetes. Limiting ROCK and arginase activity improves vascular function in diabetes.

West syndrome: response to valproate.

UNLABELLED: Management of West syndrome is unsatisfactory. In our clinic we observed that a significant proportion of patients respond to usual dose of valproate. OBJECTIVE: To prospectively assess the efficacy of valproate in controlling infantile spasms in West syndrome. METHODS: Consecutive patients presenting with West syndrome to the Pediatric Neurology Clinic or general outpatient department (OPD) were enrolled for study. Those who were not on any treatment were given valproate in a dose of 30 mg/kg/day while awaiting investigations. Patients were followed up every 2 weeks. Predefined criteria for definition of West syndrome and response were used. Those showing partial/poor response or relapse on valproate were given hormonal therapy. RESULTS: One hundred children with West syndrome were enrolled. Ninety one children were started on valproate. Of these 36 (39.5%) showed a good response, but seven later relapsed while on same dose of valproate and three were lost to follow up. Later age at onset and typical hypsarrhythmia on EEG were associated with good sustained response to valproate while a history of delayed cry at birth was associated with partial or poor response. Sixty two patients who responded poorly to or relapsed on valproate were put on hormonal treatment in addition. Of these 36 (58.1%) had a good response but 11 later relapsed after stopping treatment and two were lost to follow up. CONCLUSION: Valproate may have a role in treatment of West syndrome in a selected group of patients.

Varicella infection in a neonate with subsequent staphylococcal scalded skin syndrome and fatal shock.

A male term neonate, at day 23 of life, presented with vesicular lesions over the trunk, which spread to allover the body on the next day. Five days later, he started developing blistering of the skin over the trunk and extremities, which subsequently ruptured, leaving erythematous, tender raw areas with peeling of the skin. The mother had vesicular eruptions, which started on the second day of delivery and progressed over the next 3 days. Subsequently, similar eruptions were noticed in two of the siblings before affecting the neonate. On the basis of the exposure history and clinical picture, a diagnosis was made of varicella infection with staphylococcal scalded skin syndrome (SSSS). The blood culture and the wound surface culture grew Staphylococcus aureus. Treatment included intravenous fluid, antibiotics, acyclovir and wound care. However, after 72 h of hospitalisation, the neonate first developed shock, refractory to fluid boluses, vasopressors and catecholamine along with other supports; and he then succumbed. In all neonates, staphylococcal infection with varicella can be fatal due to SSSS, the toxic shock syndrome or septicaemia.

Physiological changes in hematological parameters during pregnancy.

Pregnancy is a state characterized by many physiological hematological changes, which may appear to be pathological in the non-pregnant state. The review highlights most of these changes along with the scientific basis for the same, as per the current knowledge, with a special reference to the red blood and white blood cells, platelets and hemostatic profile.

Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway.

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 µM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 µM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 µM) or Rho kinase (ROCK) (Y-27632, 10 µM; H1152, 0.5 µM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 µM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 µg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.

p38 Mitogen-activated protein kinase (MAPK) increases arginase activity and contributes to endothelial dysfunction in corpora cavernosa from angiotensin-II-treated mice.

INTRODUCTION: Angiotensin II (AngII) activates p38 mitogen-activated protein kinase (MAPK) and elevates arginase activity in endothelial cells. Upregulation of arginase activity has been implicated in endothelial dysfunction by reducing nitric oxide (NO) bioavailability. However, signaling pathways activated by AngII in the penis are largely unknown. AIM: We hypothesized that activation of p38 MAPK increases arginase activity and thus impairs penile vascular function in AngII-treated mice. METHODS: Male C57BL/6 mice were implanted with osmotic minipumps containing saline or AngII (42 µg/kg/h) for 14 days and cotreated with p38 MAPK inhibitor, SB 203580 (5 µg/kg/day), beginning 2 days before minipump implantation. Systolic blood pressure (SBP) was measured. Corpus cavernosum (CC) tissue was used for vascular functional studies and protein expression levels of p38 MAPK, arginase and constitutive NO synthase (NOS), and arginase activity. MAIN OUTCOME MEASURES: Arginase expression and activity; expression of phospho-p38 MAPK, endothelial NOS (eNOS) and neuronal NOS proteins; endothelium-dependent and nitrergic nerve-mediated relaxations were determined in CC from control and AngII-infused mice. RESULTS: AngII increased SBP (22%) and increased CC arginase activity and expression (∼twofold), and phosphorylated P38 MAPK levels (30%) over control. Treatment with SB 203580 prevented these effects. Endothelium-dependent NO-mediated relaxation to acetylcholine was significantly reduced by AngII and this effect was prevented by SB 203580 (P < 0.01). AngII (2 weeks) did not alter nitrergic function. However, SB 203580 significantly increased nitrergic relaxation in both control and AngII tissue at lower frequencies. Maximum contractile responses for phenylephrine and electrical field stimulation were increased by AngII (56% and 171%, respectively) and attenuated by SB 203580 treatment. AngII treatment also decreased eNOS phosphorylation at Ser-1177 compared to control. Treatment with SB 203580 prevented all these changes. CONCLUSION: p38 MAPK inhibition corrects penile arginase activity and protects against erectile dysfunction caused by AngII.

Chronic inhibition of nitric-oxide synthase induces hypertension and erectile dysfunction in the rat that is not reversed by sildenafil.

STUDY TYPE: Aetiology (case control) Level of Evidence 3b OBJECTIVE To evaluate the effect of N(G)-nitro-L-arginine methyl ester (L-NAME)-induced hypertension (HT) on erectile function in the rat and determine if the phosphodiesterase (PDE)-5 inhibitor, sildenafil, can reverse the effects of nitric oxide (NO) deficiency, as HT is a risk factor for erectile dysfunction (ED) and the NO synthase (NOS) inhibitor L-NAME induces NO-deficient HT. MATERIALS AND METHODS: Thirty-six adult Sprague-Dawley male rats were divided into three groups, i.e. a control, L-NAME-HT (40 mg/rat/day in the drinking water for 4 weeks), and sildenafil-treated L-NAME-HT (1.5 mg/rat/day sildenafil, by oral gavage concomitantly with L-NAME). The erectile response expressed as a ratio of intracavernosal pressure (ICP)/mean arterial pressure (MAP), evaluated after electrical stimulation of the right cavernous nerve. The isometric tension of corpus cavernosum smooth muscle (CCSM) was measured in organ-bath experiments. NOS expression was determined immunohistochemically for neuronal (n)NOS and by Western blot analysis for endothelial (e) and inducible (i) NOS protein. cGMP levels were evaluated by enzyme-linked immunosorbent assay. RESULTS: The erectile response was diminished in the HT group. Nitrergic and endothelium-dependent relaxation was reduced, while the relaxation response to sodium nitroprusside and contractile response to phenylephrine were not altered in CCSM from L-NAME-treated rats. HT rats showed decreased expression of nNOS, whereas eNOS and iNOS protein expression was increased. Sildenafil partly restored endothelial and molecular changes in CCSM from HT rats, but did not reverse the decreased erectile response, even as cGMP levels returned to normal levels. CONCLUSIONS: Sildenafil treatment did not correct the ED in L-NAME-treated HT rats. Under sustained high blood pressure, up-regulation of PDE5 expression failed to reverse the depletion of neuronal NO and/or impaired nNOS activity. However, endothelium-dependent relaxation was restored. Drug targeting of neuronal dysfunction might delay the onset of ED in HT.