Formulation and In Vitro-Ex vivo Evaluation of Cannabidiol and Cannabidiol-Valine-Hemisuccinate Loaded Lipid-Based Nanoformulations for Ocular Applications.

The goal of this investigation is to develop stable ophthalmic nanoformulations containing cannabidiol (CBD) and its analog cannabidiol-valine-hemisuccinate (CBD-VHS) for improved ocular delivery. Two nanoformulations, nanoemulsion (NE) and nanomicelles (NMC), were developed and evaluated for physicochemical characteristics, drug-excipient compatibility, sterilization, thermal analysis, surface morphology, ex-vivo transcorneal permeation, corneal deposition, and stability. The saturation solubility studies revealed that among the surfactants tested, Cremophor EL had the highest solubilizing capacity for CBD (23.3 ± 0.1 mg/mL) and CBD-VHS (11.2 ± 0.2 mg/mL). The globule size for the lead CBD formulations (NE and NMC) ranged between 205 and 270 nm while CBD-VHS-NMC formulation had a particle size of about 78 nm. The sterilized formulations, except for CBD-VHS-NMC at 40 °C, were stable for three months of storage (last time point tested). Release, in terms of CBD, in the in-vitro release/diffusion studies over 18 h, were faster from the CBD-VHS nanomicelles (38 %) compared to that from the CBD nanoemulsion (16 %) and nanomicelles (33 %). Transcorneal permeation studies revealed improvement in CBD permeability and flux with both formulations; however, a greater improvement was observed with the NMC formulation compared to the NE formulation. In conclusion, the nanoformulations prepared could serve as efficient topical ocular drug delivery platforms for CBD and its analog.

Anxiolytic-like effects of an mGluR 5 antagonist and a mGluR 2/3 agonist, and antidepressant-like effects of an mGluR 7 agonist in the chick social separation stress test, a dual-drug screening model of treatment-resistant depression.

Modulation of glutamate receptors has demonstrated anxiolytic and/or antidepressant effects in rodent stress models. The chick social-separation stress paradigm exposes socially raised aves to an isolation stressor which elicits distress vocalizations (DVocs) in an attempt to re-establish contact. The model presents a state of panic during the first 5 min followed by a state of behavioral despair during the last 60 to 90 min. Making it useful as a dual anxiolytic/antidepressant screening assay. Further research has identified the Black Australorp strain as a stress-vulnerable, treatment-resistant, and ketamine-sensitive genetic line. Utilizing this genetic line, we sought to evaluate modulation of glutamatergic receptors for potential anxiolytic and/or antidepressant effects. Separate dose-response studies were conducted for the following drugs: the AMPA PAM LY392098, the mGluR 5 antagonist MPEP, the mGluR 2/3 agonist LY404039, the mGluR 2/3 antagonist LY341495, and the mGluR 7 agonist AMN082. The norepinephrine α2 agonist clonidine and the NMDA antagonist ketamine were included as comparison for anxiolytic (anti-panic) and antidepressant effects, respectively. As in previous studies, clonidine reduced DVoc rates during the first 5 min (attenuation of panic) and ketamine elevated DVoc rates (attenuation of behavioral despair) during the last 60 min of isolation. The mGluR 2/3 agonist LY404039 and the mGluR 5 antagonist MPEP decreased DVoc rates during the first 5 min of isolation indicative of anxiolytic effects like that of clonidine while the mGluR 7 agonist AMN082 elevated DVoc rates in the later hour of isolation, representative of antidepressant effects like that of ketamine. Collectively, these findings suggest that certain glutamate targets may be clinically useful in treating panic disorder and/or treatment-resistant depression.

Anticancer Activity of Region B Capsaicin Analogs.

The heterocyclic vanilloid compound capsaicin is responsible for the spicy and pungent flavor of chili peppers. Several convergent studies have shown that capsaicin suppresses the growth of multiple human cancers. Apart from capsaicin, natural and synthetic capsaicin-like compounds display growth suppressive activity in human cancers. The pharmacophore of capsaicin is comprised of three regions, namely region A (the aromatic ring), region B (the amide bond), and region C (the side chain). The present manuscript describes the isolation and synthesis of capsaicin analogs which have structural modifications in region B of the molecule. Furthermore, the pharmacokinetic properties, anticancer activity of region B capsaicin analogs, as well as the signaling pathways (underlying the growth-inhibitory effects of region B capsaicin analogs) have also been described. The discovery of novel, second-generation region B capsaicin analogs may foster the hope of innovative nutrition-based combination therapies in human cancers.

Relationships between Isomeric Metabolism and Regioselective Toxicity of Hydroxychrysenes in Embryos of Japanese Medaka (Oryzias latipes).

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are ubiquitous contaminants that can be formed through oxidation of parent PAHs. Our previous studies found 2-hydroxychrysene (2-OHCHR) to be significantly more toxic to Japanese medaka embryos than 6-hydroxychrysene (6-OHCHR), an example of regioselective toxicity. We have also previously identified a sensitive developmental window to 2-OHCHR toxicity that closely coincided with liver development, leading us to hypothesize that differences in metabolism may play a role in the regioselective toxicity. To test this hypothesis, Japanese medaka embryos were treated with each isomer for 24 h during liver development (52-76 hpf). Although 6-OHCHR was absorbed 97.2 ± 0.18% faster than 2-OHCHR, it was eliminated 57.7 ± 0.36% faster as a glucuronide conjugate. Pretreatment with cytochrome P450 inhibitor, ketoconazole, reduced anemia by 96.8 ± 3.19% and mortality by 95.2 ± 4.76% in 2-OHCHR treatments. Formation of chrysene-1,2-diol (1,2-CAT) was also reduced by 64.4 ± 2.14% by ketoconazole pretreatment. While pretreatment with UDP-glucuronosyltransferase inhibitor, nilotinib, reduced glucuronidation of 2-OHCHR by 52.4 ± 2.55% and of 6-OHCHR by 63.7 ± 3.19%, it did not alter toxicity for either compound. These results indicate that CYP-mediated activation, potentially to 1,2-CAT, may explain the isomeric differences in developmental toxicity of 2-OHCHR.

Stage Dependent Enantioselective Metabolism of Bifenthrin in Embryos of Zebrafish (Danio rerio) and Japanese Medaka (Oryzias latipes).

Bifenthrin (BF) is a widely used pyrethroid that has been frequently detected in surface waters. Previous studies indicated that BF had antiestrogenic activity in zebrafish embryos but estrogenic activity in posthatch fish. To determine whether age-related differences in metabolism contribute to the endocrine effects in developing fish, embryos from zebrafish and Japanese medaka were exposed to BF before and after liver development. Since the commercial mixture of BF is an isomer-enriched product containing two enantiomers (1R-cis-BF and 1S-cis-BF), enantioselective metabolism was also evaluated. The estrogenic metabolite, 4-hydroxybifenthrin (4-OH-BF) was identified in zebrafish embryos, and formation was higher in animals after liver development (>48 hpf). Treatments with ß-glucuronidase indicated that 4-OH-BF underwent conjugation in embryos. Formation was reduced by cotreatment of the cytochrome P450 (CYP450) inhibitor, ketoconazole. Formation of 4-OH-BF was greater when treated with 1R-cis-BF compared to the S-enantiomer. However, metabolites were not observed in medaka embryos. These data indicate enantioselective oxidation of BF to an estrogenic metabolite occurs in zebrafish embryos and, since it is increased after liver development, may partially explain estrogenic activity observed in older animals. The lack of activity in medaka suggests species-specific effects with BF metabolism and may influence risk assessment strategies in wildlife.

Self-Organized Amphiphiles Are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments.

Analysis of membrane protein topography using fast photochemical oxidation of proteins (FPOP) has been reported in recent years but is still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. We found no significant change in bulk solvent radical scavenging activity upon the addition of disrupted cellular membranes up to 25600 cells/µL using an inline radical dosimeter. We confirmed the nonscavenging nature of the membrane in bulk solution with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration is a potent hydroxyl radical scavenger, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. Examination of both an extracellular peptide of the integral membrane protein bacteriorhodopsin as well as a novel hydroxyl radical dosimeter tethered to a Triton X-series amphiphile indicate that proximity to the membrane surface greatly decreases reaction with hydroxyl radicals, even though the oxidation target is equally solvent accessible. These results suggest that the observed reduced oxidation of solvent-accessible surfaces of integral membrane proteins is due to the high local concentration of radical scavengers in the membrane or membrane mimetics competing for the local concentration of hydroxyl radicals.

Nonpungent N-AVAM Capsaicin Analogues and Cancer Therapy.

Capsaicin displays robust growth-inhibitory activity in multiple human cancers. However, the feasibility of capsaicin as a clinically relevant anticancer drug is hampered by its adverse side effects. This concern has led to extensive research focused on the isolation and synthesis of second-generation nonpungent capsaicin analogues with potent antineoplastic activity. A major class of nonpungent capsaicin-like compounds belongs to the N-acyl-vanillylamide (N-AVAM) derivatives of capsaicin (hereafter referred as N-AVAM capsaicin analogues). This perspective discusses the isolation of N-AVAM capsaicin analogues from natural sources as well as their synthesis by chemical and enzymatic methods. The perspective describes the pharmacokinetic properties and anticancer activity of N-AVAM capsaicin analogues. The signaling pathways underlying the growth-inhibitory effects of N-AVAM capsaicin analogues have also been highlighted. It is hoped that the insights obtained in this perspective will facilitate the synthesis of a second generation of N-AVAM capsaicin analogues with improved stability and growth-suppressive activity in human cancer.

Novel Disinfection Byproducts Formed from the Pharmaceutical Gemfibrozil Are Bioaccumulative and Elicit Increased Toxicity Relative to the Parent Compound in Marine Polychaetes (Neanthes arenaceodentata).

Formation of halogenated disinfection byproducts (DBPs) from pharmaceutically active compounds has been observed in water supply systems following wastewater chlorination. Although research has been limited thus far, several studies have shown that halogenated DBPs may elicit increased toxicity compared to their parent compounds. For example, the lipid regulator gemfibrozil has been shown to form chlorogemfibrozil (Cl-gemfibrozil) and bromogemfibrozil (Br-gemfibrozil) following chlorination, which are more potent antiandrogens in male Japanese medaka (Oryzias latipes) compared to their parent compounds. In the present study, we aimed to characterize the bioaccumulative ability of halogenated gemfibrozil DBPs in marine polychaetes via chronic sediment exposures and, consequently, to assess the chronic and acute toxicity of halogenated gemfibrozil DBPs through sediment (in vivo) and aqueous (in vivo and in silico) toxicity evaluations. Following 28 day sediment exposures, Cl-gemfibrozil and Br-gemfibrozil bioaccumulated within Neanthes arenaceodentata, with both compounds reducing survival and growth. The biota-sediment accumulation factors determined for Cl-gemfibrozil and Br-gemfibrozil were 2.59 and 6.86, respectively. Furthermore, aqueous 96 h toxicity tests with N. arenaceodentata indicated that gemfibrozil DBPs elicited increased toxicity compared to the parent compound. While gemfibrozil had an acute LC50 value of 469.85 ± 0.096 mg/L, Cl-gemfibrozil and Br-gemfibrozil had LC50 values of 12.34 ± 0.085 and 9.54 ± 0.086 mg/L, respectively. Although acute toxicity is relatively low, our results indicate that halogenated gemfibrozil DBPs are bioaccumulative and may elicit effects in apex food web organisms prone to accumulation following lifelong exposures.

Structure-Based Identification of Potent Natural Product Chemotypes as Cannabinoid Receptor 1 Inverse Agonists.

Natural products are an abundant source of potential drugs, and their diversity makes them a rich and viable prospective source of bioactive cannabinoid ligands. Cannabinoid receptor 1 (CB1) antagonists are clinically established and well documented as potential therapeutics for treating obesity, obesity-related cardiometabolic disorders, pain, and drug/substance abuse, but their associated CNS-mediated adverse effects hinder the development of potential new drugs and no such drug is currently on the market. This limitation amplifies the need for new agents with reduced or no CNS-mediated side effects. We are interested in the discovery of new natural product chemotypes as CB1 antagonists, which may serve as good starting points for further optimization towards the development of CB1 therapeutics. In search of new chemotypes as CB1 antagonists, we screened the in silico purchasable natural products subset of the ZINC12 database against our reported CB1 receptor model using the structure-based virtual screening (SBVS) approach. A total of 18 out of 192 top-scoring virtual hits, selected based on structural diversity and key protein⁻ligand interactions, were purchased and subjected to in vitro screening in competitive radioligand binding assays. The in vitro screening yielded seven compounds exhibiting >50% displacement at 10 µM concentration, and further binding affinity (Ki and IC50) and functional data revealed compound 16 as a potent and selective CB1 inverse agonist (Ki = 121 nM and EC50 = 128 nM) while three other compounds-2, 12, and 18-were potent but nonselective CB1 ligands with low micromolar binding affinity (Ki). In order to explore the structure⁻activity relationship for compound 16, we further purchased compounds with >80% similarity to compound 16, screened them for CB1 and CB2 activities, and found two potent compounds with sub-micromolar activities. Most importantly, these bioactive compounds represent structurally new natural product chemotypes in the area of cannabinoid research and could be considered for further structural optimization as CB1 ligands.

Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays will be important for many aspects of collection, transport, medical treatment in aquaria and conservation management of these charismatic and ecologically important species.

Heme oxygenase-1 is a potent inhibitor of placental ischemia-mediated endothelin-1 production in cultured human glomerular endothelial cells.

Preeclampsia is a pregnancy-specific disorder of maternal hypertension and reduced renal hemodynamics linked to reduced endothelial function. Placental ischemia is thought to be the culprit of this disease, as it causes the release of factors like tumor necrosis factor (TNF)-α that induce vascular endothelin-1 (ET-1) production. Interestingly, placental ischemia-induced hypertension in rats [reduced uterine perfusion pressure (RUPP) model] is abolished by ETA receptor blockade, suggesting a critical role for ET-1. Although it has been found that systemic induction of heme oxygenase (HO)-1 is associated with reduced ET-1 production and attenuated hypertension, it is unclear whether HO-1 directly modulates the increased ET-1 response to placental factors. We tested the hypothesis that HO-1 or its metabolites inhibit ET-1 production in human glomerular endothelial cells induced by serum of RUPP rats or TNF-α. Serum (5%) from RUPP hypertensive (mean arterial blood pressure 119 ± 9 mmHg) vs. normotensive pregnant (NP, 101 ± 6 mmHg, P < 0.001) rats increased ET-1 production (RUPP 168.8 ± 18.1 pg/ml, NP 80.3 ± 22.7 pg/ml, P < 0.001, n = 12/group). HO-1 induction [25 µM cobalt photoporphyrin (CoPP)] abolished RUPP serum-induced ET-1 production (1.6 ± 0.8 pg/ml, P < 0.001), whereas bilirubin (10 µM) significantly attenuated ET-1 release (125.3 ± 5.2 pg/ml, P = 0.005). Furthermore, TNF-α-induced ET-1 production (TNF-α 31.0 ± 8.4 vs. untreated 7.5 ± 0.4 pg/ml, P < 0.001) was reduced by CoPP (1.5 ± 0.8 pg/ml, P < 0.001) and bilirubin (10.5 ± 4.3 pg/ml, P < 0.001). These results suggest that circulating factors released during placental ischemia target the maternal glomerular endothelium to increase ET-1, and that pharmacological induction of HO-1 or bilirubin could be a treatment strategy to block this prohypertensive pathway in preeclampsia.

Carbon Monoxide Releasing Molecules Blunt Placental Ischemia-Induced Hypertension.

BACKGROUND: Preeclampsia is a pregnancy complication which manifests as new-onset hypertension, proteinuria, and a spectrum of other symptoms. While the underlying causes are still a subject of much debate, it is commonly believed that placental ischemia is a central cause. The ischemic placenta secretes factors which are believed to be responsible for the maternal syndrome; most notably the anti-angiogenic protein soluble fms-like tyrosine kinase 1 (sFlt-1). We have reported that induction of the carbon monoxide (CO) producing protein heme oxygenase-1 restored angiogenic imbalance and reduced blood pressure in a rat model of placental ischemia, and that CO blocks hypoxia-induced sFlt-1 production from placental tissue in vitro. We therefore hypothesized that direct administration of CO by a CO-releasing molecule (CORM) would blunt the placental ischemia-induced increase in sFlt-1 and thus the hypertension characteristic of this model. METHODS: We administered a soluble CO donor molecule (CORM-3) daily i.v. in control animals or those undergoing placental ischemia from GD14. Blood pressure and renal function were measured on GD19, and angiogenic markers measured by ELISA. RESULTS: Interestingly, though we found that CORM administration significantly blunted the hypertensive response to placental ischemia, there was no concomitant normalization of sFlt-1 in either the placenta or maternal circulation. We did find, however, that CORM administration caused a significant increase in glomerular filtration rate, presumably by vasodilation of the renal arteries and increased renal plasma flow. CONCLUSIONS: All in all these data suggest that administration of CO by CORMs do lower blood pressure during placental ischemia mechanisms independent of changes in angiogenic balance.

Biochemical Mechanisms for Geographical Adaptations to Novel Toxin Exposures in Butterflyfish.

Some species of butterflyfish have had preyed upon corals for millions of years, yet the mechanism of butterflyfish specialized coral feeding strategy remains poorly understood. Certain butterflyfish have the ability to feed on allelochemically rich soft corals, e.g. Sinularia maxima. Cytochrome P450 (CYP) is the predominant enzyme system responsible for the detoxification of dietary allelochemicals. CYP2-like and CYP3A-like content have been associated with butterflyfish that preferentially consumes allelochemically rich soft corals. To investigate the role of butterflyfish CYP2 and CYP3A enzymes in dietary preference, we conducted oral feeding experiments using homogenates of S. maxima and a toxin isolated from the coral in four species of butterflyfish with different feeding strategies. After oral exposure to the S. maxima toxin 5-episinulaptolide (5ESL), which is not normally encountered in the Hawaiian butterflyfish diet, an endemic specialist, Chaetodon multicinctus experienced 100% mortality compared to a generalist, Chaetodon auriga, which had significantly more (3-6 fold higher) CYP3A-like basal content and catalytic activity. The specialist, Chaetodon unimaculatus, which preferentially feed on S. maxima in Guam, but not in Hawaii, had 100% survival, a significant induction of 8-12 fold CYP3A-like content, and an increased ability (2-fold) to metabolize 5ESL over other species. Computer modeling data of CYP3A4 with 5ESL were consistent with microsomal transformation of 5ESL to a C15-16 epoxide from livers of C. unimaculatus. Epoxide formation correlated with CYP3A-like content, catalytic activity, induction, and NADPH-dependent metabolism of 5ESL. These results suggest a potentially important role for the CYP3A family in butterflyfish-coral diet selection through allelochemical detoxification.

Adaptation of a corticosterone ELISA to demonstrate sequence-specific effects of angiotensin II peptides and C-type natriuretic peptide on 1alpha-hydroxycorticosterone synthesis and steroidogenic mRNAs in the elasmobranch interrenal gland.

It is thought that a single corticosteroid, 1alpha-hydroxycorticosterone (1alpha-B), is both a glucocorticoid and mineralocorticoid in the elasmobranch fishes. We investigated the putative mineralocorticoid role of 1alpha-B by examining regulation of interrenal 1alpha-B synthesis by osmoregulatory hormones in the euryhaline stingray Dasyatis sabina. Using synthesized steroid, a commercial enzyme-linked immunoassay was validated for the quantification of 1alpha-B. In interrenal cultures, the antinatriuretic peptide angiotensin II (ANG II) was potently steroidogenic, whereas C-type natriuretic peptide had no effect on 1alpha-B titers. However, both peptides significantly decreased abundance of rate-limiting steroidogenic mRNAs (steroidogenic acute regulatory protein, StAR; cholesterol side-chain cleavage, P450scc). We also isolated cDNAs encoding ANG II from three species of elasmobranch, verifying heterogeneity among elasmobranch peptides at the first amino acid position. Potential implications of this heterogeneity were investigated by examining the effects of homologous and heterologous ANG II on interrenal steroid production and steroidogenic mRNAs. Changes at amino acid position three, but not position one, of ANG II significantly affected steroidogenic potency. Conversely, changes at position one, but not position three, significantly affected the potency of ANG II to alter levels of steroidogenic mRNAs. This study is the first to demonstrate regulation of elasmobranch steroidogenic mRNAs by osmoregulatory peptides.

In vivo inhibition of renal heme oxygenase with an imidazole-dioxolane inhibitor.

Recent studies have identified imidazole-dioxolane based compounds as novel heme oxygenase (HO) inhibitors. While these compounds have been demonstrated to be specific HO inhibitors in vitro, they have yet to be used to inhibit renal HO activity in vivo. The goal of this study was to determine the effectiveness of the imidazole-dioxolane HO-1 inhibitor, QC-13, in the inhibition of renal HO activity in vivo. HO-1 was induced in mice by treatment with cobalt protoporphyrin (CoPP). After 5 days, QC-13 was delivered either by continuous intrarenal medullary interstitial infusion (IRMI) into one kidney at several concentrations for 72 h or by two intraperitoneal injections over a 48-h period. IRMI infusion of QC-13 at a concentration of 25 microM resulted in a significant decrease in medullary but not cortical HO activity as compared to CoPP treated kidneys. IRMI infusion of QC-13 at a lower concentration (2.5 microM) had no effect on either medullary or cortical HO activity in CoPP treated mice. In contrast, administration of QC-13 at a higher concentration (250 microM) resulted in a significant decrease in both medullary and cortical HO activity in CoPP treated mice. Systemic administration of QC-13 resulted in significant decrease both renal cortical and medullary HO activity in CoPP treated mice. In contrast to classical porphyrin based HO inhibitors, IRMI infusion of QC-13 did not induce HO-1 protein levels as determined by Western blot analysis of medullary protein samples. Our results demonstrated that imidazole-dioxolane inhibitors are renal HO inhibitors in vivo and can inhibit HO activity independent of HO-1 induction. These inhibitors may be useful tools to elucidate the role of renal HO-1 in numerous physiologic and pathophysiologic conditions.

Heme oxygenase attenuates angiotensin II-mediated superoxide production in cultured mouse thick ascending loop of Henle cells.

Heme oxygenase (HO)-1 induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is not clear. The goal of this study was to test the hypothesis that induction of HO-1 can reduce the ANG II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH (mTALH) cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 microM) or hemin (50 microM) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with 10(-9) M ANG II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5+/-5 to 136+/-18 relative fluorescence units (RFU)/microm2. Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced ANG II-induced DHE fluorescence to 64+/-5, 64+/-8, and 41+/-4 RFU/microm2, respectively. To determine which metabolite of HO-1 is responsible for reducing ANG II-mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or carbon monoxide (CO)-releasing molecule (CORM)-A1 (each at 100 microM) before exposure to ANG II. DHE fluorescence averaged 80+/-7 RFU/microm2 after incubation with ANG II and was significantly decreased to 55+/-7 and 53+/-4 RFU/microm2 after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces the levels of ANG II-mediated superoxide production through the production of both bilirubin and CO.

Heme oxygenase-1 induction does not improve vascular relaxation in angiotensin II hypertensive mice.

BACKGROUND: Induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II)-dependent hypertension in mice. However, the mechanism by which HO-1 lowers blood pressure in this model is not clear. This study was designed to determine whether induction of HO-1 results in an improvement in vascular relaxation in Ang II hypertensive mice. METHODS: Mice were treated with either of the vehicles (control), the HO-1 inducer cobalt protoporphyrin (CoPP;50 mg/kg), Ang II(1 microg/kg/min, 14 days), or Ang II + CoPP. CoPP was administered as a single bolus dose 2 days prior to subcutaneous implantation of the osmotic minipump containing Ang II. Vascular relaxation was examined in isolated carotid arteries precontracted with the thromboxane mimetic U46619 (0.4 microg/ml). RESULTS: Endothelial dependent relaxation to acetylcholine (ACh; 1 micromol/l) was significantly impaired in Ang II-treated mice compared to control mice (56 +/- 3% vs. 40 +/- 4%, P < 0.05, n > or = 6). Similarly, endothelial independent relaxation to sodium nitroprusside (SNP; 1 micromol/l) was significantly impaired in Ang II mice (56 +/- 6% vs. 28 +/- 6%, P < 0.05, n > or = 6). Relaxation in response to the carbon monoxide donor, CORM-A1 (100 micromol/l), was attenuated after Ang II treatment (75 +/- 7% vs. 59 +/- 7%,P < 0.05, n > or = 6). CoPP treatment induced HO-1 but not HO-2 protein in the aorta, as measured by western blot analysis. CoPP treatment had no effect on vascular responses in control mice and did not improve ACh (26 +/- 5%, n = 15), SNP (23 +/- 4%, n = 15), or CORM-A1 (46 +/- 7%, n = 10) dependent relaxation in Ang II treated mice. CONCLUSIONS: These results suggest that induction of HO-1 lowers Ang II-dependent hypertension through a mechanism independent of improved vascular relaxation.

Renal vascular responses to CORM-A1 in the mouse.

CORM-A1 is a newly described water-soluble carbon monoxide (CO) releasing molecule (CORM) that can deliver CO to various vascular beds in the absence of dramatic changes in blood carboxy-hemoglobin (COHb) levels. We tested the in vivo and in vitro renal vascular effects of CORM-A1 administration using anesthetized mice instrumented with a renal flow probe as well as in isolated, pressurized renal interlobar arteries. Administration of CORM-A1 (0.96 micromol) resulted in a significant increase in renal blood flow (RBF) of 33 +/- 6% as compared to control. Administration of acetylcholine (50 pmol) caused a similar increase in RBF (25 +/- 4%). In order to determine if the vasodilatory effect of CORM-A1 in vivo was mediated through activation of soluble guanylate cyclase (sGC), mice were pretreated with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 nmolkg(-1)min(-1)) for 30min. Pretreatment with ODQ significantly reduced the CORM-A1 mediated increase in RBF to 9 +/- 5% of control. In isolated pressurized renal interlobar arteries, CORM-A1 caused dose dependent vasodilatation of phenylephrine constricted arteries. The CORM-A1 mediated vasodilatation was significantly attenuated by ODQ to similar levels as observed in vivo. Inhibition of calcium activated potassium channels (Kca) with iberiotoxin resulted in a complete blockade of the CORM-A1 mediated vasodilatation in pressurized renal interlobar arteries. We conclude that CO released from CORM-A1 causes an increase in RBF and a decrease in vascular resistance through activation of sGC and opening of Kca channels in the kidney of the mouse.