Aortic Strips Research Articles

Possible Involvement of Insulin and Oxidative Stress in Vascular Dysfunction of Diabetic Mellitus

Macro- and microvascular disease states currently represent the principal causes of morbidity and mortality in patients with type I or type II diabetes mellitus. Abnormal vasomotor responses and impaired endothelium-dependent vasodilation have been demonstrated in various beds in different animal models of diabetes and in humans with type I or type II diabetes. The principal mediators of diabetes-associated vascular dysfunction are increases in glucose, oxidized low-density lipoprotein, endothelin-1, angiotensin II, insulin, or growth factors. An accumulating body of evidence indicates that abnormal production of oxidative stress may be one of several factors contributing to vascular dysfunction in diabetes. It is possible that in diabetic states, hyperinsulinemia initiates oxidant stress, leading to vascular dysfunction at a later stage. We and others have demonstrated that in models of hyperinsulinemia and hyperglycemia, . NO production and/or . NO responsiveness are impaired in aortic strips. Several recent studies have shown that the formation of nitrotyrosine and/or peroxynitrite impairs vascular . NO responsiveness and . NO production. Our findings suggest that the coexistence of a high insulin level and an established diabetic state may lead to the excessive generation of peroxynitrite, and that this may in turn trigger an impairment of endothelium-dependent relaxation via a decrease in sarcoendo plasmic reticulum Ca(2+) ATPase function. This review summarizes the results of our recent studies on the involvement of insulin and oxidative stress in the blood vessels of diabetic animals.

Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

Epinephrine, through its action on β-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca2+ ([Ca2+]i) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca2+ influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca2+ dye, it was found that epinephrine and isoprenaline, a β-adrenoceptor agonist, induced endothelial Ca2+ influx in the presence of bradykinin. This Ca2+ influx was inhibited by L-cis-diltiazem and LY-83583, and by a β2-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca2+ influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca2+ influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate β-adrenoceptor agonist-induced endothelial Ca2+ influx and subsequent vascular dilation.

Nitric oxide pathway-mediated relaxant effect of aqueous sesame leaves extract (Sesamum radiatum Schum. & Thonn.) in the guinea-pig isolated aorta smooth muscle

BackgroundSesamum radiatum Schum. & Thonn. (Pedaliaceae) is an annual herbaceous plant, which belongs to the family Pedaliaceae and genus Sesamum. Sesame is used in traditional medicine in Africa and Asia for many diseases treatment. Sesame plant especially the leaves, seed and oil are consumed locally as a staple food by subsistence farmers. The study analyses the relaxation induced by the aqueous extract of leaves from sesame (ESera), compared with those of acetylcholine (ACh) in the guinea-pig aortic preparations (GPAPs), in order to confirm the use in traditional medicine for cardiovascular diseases.MethodsThe longitudinal strips of aorta of animals were rapidly removed from animals. The aorta was immediately placed in a Mac Ewen solution. Experiments were performed in preparations with intact endothelium as well as in aortae where the endothelium had been removed. The preparations were suspended between two L-shaped stainless steel hooks in a 10 ml organ bath with Mac Ewen solution. The isometric contractile force of the aorta strips of guinea-pig were recorded by using a strain gauge. All both drugs caused concentration-dependent relaxations responses.ResultsThe aqueous extract of leaves from sesame ESera (1 × 10-7 – 0.1 μg/ml) caused a graded relaxation in GPAPs with intact endothelium, with a EC50-value of 1 × 10-4 μg/ml. The same effect was observed with ACh (7 × 10-2 nM – 7 × 10-1 μM), which caused relaxation in a concentration-dependent manner. The relaxation in response to ESera and, like that to ACh in GPAPs without endothelium, was fully abolished. Destruction of the endothelium or incubation with the nitric oxyde synthase inhibitor (L-NNA) significantly enhanced the inhibition of the relaxation response to ESera. Moreover, all concentrations induced vasoconstrictions. However, L-NNA produced a significant displacement to the right (about 65-fold) of the relaxation response to ESera. Similar results were obtained with ACh. Both diclofenac and tetra-ethyl-ammonium (TEA) pretreatment of GPAPs induced a suppression of the relaxation caused by ESera, and produced a very significant rightward shifts of the CRC (16-fold) for diclofenac and increase the Emax. In contract, the relaxation caused by ACh was not significantly affected by diclofenac or by TEA.ConclusionThus, the present results indicate clearly that the nitric oxide largely contribute to the relaxation effect of Esera and of ACh in GPAPs. In addition, their contractile effects are also mediated by cyclooxygenase activation, and probably the K+ channels involvement, that confirm the use of various preparations of Esera for the treatments of cardiovascular diseases.

Serotonergic Properties of the Roots of Clerodendron capitatum

The aim of this study is to investigate the pharmacological effects of the methanolic extract of Clerodendron capitatum, CC, (family verbenaceae). These investigations were carried out on isolated frog rectus abdominus muscle and rabbit aortic strips and jejunum and non-pregnant rat uterus pretreated with oestradiol. Results showed that CC did not demonstrate any contracting, relaxant or blocking effects on frog rectus abdominus muscle and rat aortic strip preparations. The extract produced a concentration-dependent (p<0.05, t-test, n = 6) decrease of the normal rhythmic contraction of rabbit jejunum, however, this effect was reversed by prior addition of cyproheptadine (non-specific 5-HT antagonist). In addition, CC also produced a stimulant activity on rat uterus which was blocked by cyproheptadine. Future study is recommended to study the specificity of CC to different 5-HT receptors by using different isolated animal tissues and specific 5-HT antagonists.

Attenuation of maitotoxin-induced cytotoxicity in rat aortic smooth muscle cells by inhibitors of Na +/Ca 2+ exchange, and calpain activation

The highly potent marine toxin maitotoxin (MTX) evoked an increase in cytosolic Ca 2+ levels in fura-2 loaded rat aortic smooth muscle cells, which was dependent on extracellular Ca 2+. This increase was almost fully inhibited by KB-R7943, a potent selective inhibitor of the reverse mode of the Na +/Ca 2+ exchanger (NCX). Cell viability was assessed using ethidium bromide uptake and the alamarBlue cytotoxicity assay. In both assays MTX-induced toxicity was attenuated by KB-R7943, as well as by MDL 28170, a membrane permeable calpain inhibitor. Maitotoxin-evoked contractions of rat aortic strip preparations in vitro, which persist following washout of the toxin, were relaxed by subsequent addition of KB-R7943 or MDL 28170, either in the presence of, or following washout of MTX. These results suggest that MTX targets the Na +/Ca 2+ exchanger and causes it to operate in reverse mode (Na + efflux/Ca 2+ influx), thus leading to calpain activation, NCX cleavage, secondary Ca 2+ overload and cell death.

Design, Synthesis, and Biological Evaluation of AT1 Angiotensin II Receptor Antagonists Based on the Pyrazolo[3,4-b]pyridine and Related Heteroaromatic Bicyclic Systems

Novel AT 1 receptor antagonists bearing the pyrazolo[3,4- b]pyridine bicyclic heteroaromatic system (or structurally related moieties) were designed and synthesized as the final step of a large program devoted to the development of new antihypertensive agents and to the understanding of the molecular basis of their pharmacodynamic and pharmacokinetic properties. The preliminary pharmacological characterization revealed nanomolar AT 1 receptor affinity for several compounds of the series and a potent antagonistic activity in isolated rabbit aortic strip functional assay for 7c and 8a. These results stimulated the study of the biopharmaceutical properties of some selected compounds, which were found to be characterized by a permeability from medium to high. Remarkably, the least permeable 7c showed both permeability and oral bioavailability (80%) higher than losartan, but its terminal half-life was shorter. These results suggest that the permeability is not a limiting factor in the pharmacokinetics of these AT 1 receptor antagonists.

Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with 14C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid-sensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA; 10(-6) M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10(-7)-10(-4) M) and acetylcholine (10(-9)-10(-6) M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K(+)] from 4.8 to 20 mM. AA (3 x 10(-6) M) induced cell membrane hyperpolarization (from -31.0 +/- 1 to -46.8 +/- 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor.

Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli

To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant human urodilatin, a hormone for the treatment of acute decompensated heart failure. The fusion protein, which was overexpressed mainly as inclusion bodies in Escherichia coli, constituted about 25% of the total cell proteins. After purification by Ni-sepharose affinity chromatography and renaturation in refolding buffer, the fusion protein was cleaved with SUMO protease 1. Urodilatin was separated from the fusion partner by the subtractive chromatography using Ni-sepharose once again, and then further purified with reverse-phase high performance liquid chromatography. In vitro activity assay demonstrated that the recombinant urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.77+/-0.53 microg/ml, which was similar to that of the synthetic urodilatin standard. The expression strategy presented in this study allows convenient high yield and easy purification of small recombinant peptides with native sequences.

5-Hydroxytryptamine Lowers Blood Pressure in Normotensive and Hypertensive Rats

Arterial hyper-responsiveness to 5-hydroxytryptamine (5-HT) is a hallmark of hypertension, and plasma levels of free 5-HT are elevated in hypertension. We hypothesized that chronic administration of 5-HT would cause blood pressure to 1) rise in normotensive rats and 2) rise significantly more in hypertensive rats. The deoxycorticosterone acetate (DOCA)-salt hypertensive and sham normotensive rat were used. Animals were implanted with minipumps that delivered 5-HT (or vehicle) at a rate of 25 microg/kg/min for 7 days. Free plasma 5-HT was elevated significantly by this protocol. Within 48 h, mean arterial blood pressure measured telemetrically decreased in sham (106 +/- 2 to 83 +/- 2 mm Hg) and in DOCA-salt hypertensive (166 +/- 9 to 112 +/- 3 mm Hg) rats; vehicle did not change blood pressure in either group. Ganglionic blockade (hexamethonium) reduced blood pressure to a greater magnitude in DOCA vehicle-administered rats (peak fall arterial pressure, 91 +/- 14 mm Hg) compared with DOCA 5-HT-administered rats (40 +/- 6 mm Hg). Maximal acetylcholine-induced (NO-dependent) relaxation in phenylephrine-contracted aortic strips was greater in 5-HT-administered (69.2 +/- 9.1% relaxation) versus vehicle-administered (39.7 +/- 14.2%) DOCA rats; aortic endothelial cell nitric oxide synthase expression was higher in the 5-HT- versus vehicle-administered DOCA-salt rats. In normotensive and DOCA-salt hypertensive rats, the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine (0.5 g/l in water) prevented the fall in blood pressure to 5-HT. We conclude that chronic exogenous 5-HT reduces blood pressure in normotensive and hypertensive rats through mechanisms critically dependent on NOS.

Searching for New NO-Donor Aspirin-like Molecules: A New Class of Nitrooxy-acyl Derivatives of Salicylic Acid

A new class of products in which the phenol group of salicylic acid is linked to alkanoyl moieties bearing nitrooxy functions has been synthesized and studied for their polyvalent actions. The products were stable in acid and neutral media, while they were hydrolyzed in human serum. Their half-lives were dependent upon the structure of alkanoyl moieties. The products showed anti-inflammatory activities similar to aspirin when tested in the carrageenan-induced paw edema assay in the rat. Interestingly, unlike aspirin, they showed reduced or no gastrotoxicity in a lesion model in rats at equimolar doses. A number of them were able to inhibit platelet aggregation induced by collagen in human platelet-rich plasma. All of the products were capable of relaxing rat aortic strips precontracted with phenylephrine in a concentration-dependent manner. Selected members of this new class of nonsteroidal anti-inflammatory drugs might represent possible safer alternatives to aspirin in different clinical settings.

CNGA2 Channels Mediate Adenosine-Induced Ca 2+ Influx in Vascular Endothelial Cells

Adenosine is a cAMP-elevating vasodilator that induces both endothelium-dependent and -independent vasorelaxation. An increase in cytosolic Ca(2+) ([Ca(2+)](i)) is a crucial early signal in the endothelium-dependent relaxation elicited by adenosine. This study explored the molecular identity of channels that mediate adenosine-induced Ca(2+) influx in vascular endothelial cells. Adenosine-induced Ca(2+) influx was markedly reduced by L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated (CNG) channels, in H5V endothelial cells and primary cultured bovine aortic endothelial cells (BAECs). The Ca(2+) influx was also inhibited by 2 adenylyl cyclase inhibitors MDL-12330A and SQ-22536, and by 2 A(2B) receptor inhibitors MRS-1754 and 8-SPT, but not by an A(2A) receptor inhibitor SCH-58261 or a guanylyl cyclase inhibitor ODQ. Patch clamp experiments recorded an adenosine-induced current that could be inhibited by L-cis-diltiazem and LY-83583. A CNGA2-specific siRNA markedly decreased the Ca(2+) influx and the cation current in H5V cells. Furthermore, L-cis-diltiazem inhibited the endothelial Ca(2+) influx in mouse aortic strips, and it also reduced 5-N-ethylcarboxamidoadenosine (NECA, an A(2) adenosine receptor agonist)-induced vasorelaxation. CNGA2 channels play a key role in adenosine-induced endothelial Ca(2+) influx and vasorelaxation. It is likely that adenosine acts through A(2B) receptors and adenylyl cyclases to stimulate CNGA2.

Evidence Mounts That Nitrite Contributes to Hypoxic Vasodilation in the Human Circulation

Hypoxic vasodilation is a conserved physiological response to hypoxia that matches blood flow and oxygen delivery to tissue metabolic demand. This fundamental physiological process has been characterized for >100 years since the initial description by Roy and Brown1 in 1880. Hypoxic vasodilation requires a sensor mechanism that can detect a divergence in the normal relationship between delivered blood oxygen and tissue oxygen consumption.2 This hypoxic sensor mechanism must be coupled to the feedback generation of vasodilatory effectors that increase blood flow to maintain adequate tissue oxygenation. In short, hypoxic vasodilation requires hypoxia and/or pH sensing coupled to the release of a vasodilating signal. In mammalian species, the set point for hypoxic vasodilation occurs as the hemoglobin desaturates from 60% to 40%, around a partial pressure of oxygen ranging from 40 to 20 mm Hg.3 Despite the fact that the hypoxic vasodilation response was discovered almost 150 years ago, the identities of the oxygen sensor mechanism and the specific feedback vasodilator effectors remain uncertain. Although a number of mediators have been considered, including adenosine, nitric oxide (NO), ATP-sensitive potassium (KATP) channels, endothelium-derived hyperpolarizing factor (candidates include CO, H2O2, or ONOO−), and prostacyclin,2,4 the specific blockade of many of these pathways fails to completely inhibit hypoxic vasodilation.2 Article p 670 The present study published by Maher and colleagues5 in this issue of Circulation provides compelling evidence in normal human volunteers that the circulating anion salt nitrite (NO2−) may be an effector of hypoxic vasodilation. They report that nitrite potently vasodilates the venous forearm circulation under resting conditions and potently vasodilates the arteriolar circulation during experimental hypoxia, achieved by systemic breathing of 11% oxygen. Their studies indicate that the vasodilatory effect of nitrite is maximal in the deoxygenated venous …

Ascorbic Acid Decreases the Binding Affinity of the AT1 Receptor for Angiotensin II

Ascorbic acid is an essential vitamin and a powerful antioxidant. Many studies have highlighted the benefits of ascorbic acid for chronic cardiovascular diseases such as hypertension in which angiotensin II (Ang II) plays an significant role. We therefore hypothesized that ascorbic acid could modify the pharmacological properties of the AT(1) receptor for Ang II. Binding studies and Ca(2+) mobilization studies were performed with HEK293 cells stably expressing the AT(1) receptor for Ang II. Smooth muscle contraction studies were performed with rabbit aorta strips that endogenously express the AT(1) receptor. Scatchard analysis revealed that ascorbic acid decreased the binding affinity of the AT(1) receptor without modifying its maximal binding capacity. Ascorbic acid did not modify the binding affinity of the AT(2) receptor for Ang II or of the UT receptor for urotensin II. In single-cell Ca(2+) imaging assays, ascorbic acid reduced the frequency of intracellular Ca(2+) oscillations induced by a low dose of Ang II. In functional assays, ascorbic acid significantly diminished the contraction of rabbit aorta pre-contracted with Ang II but not those pre-contracted with urotensin II. Ascorbic acid decreases the binding affinity of the AT(1) receptor. These results offer a mechanistic explanation for the reported blood pressure lowering effect of ascorbic acid.

Nitric Oxide Donor β2-Agonists: Furoxan Derivatives Containing the Fenoterol Moiety and Related Furazans

The structure of fenoterol, a beta2-adrenoceptor agonist used in therapy, has been joined with furoxan NO-donor moieties to give new NO-donor beta2-agonists. The furazan analogues, devoid of the property to release NO, were also synthesized for comparison. All the compounds retained beta2-agonistic activity at micromolar or submicromolar concentration when tested on guinea pig tracheal rings precontracted with carbachol. Among the furoxan derivatives, the NO contribution to trachea relaxation was evident with product 15b at micromolar concentrations. All the new NO-donor hybrids were able to dilate rat aortic strips precontracted with phenylephrine. Both furoxan and furazan derivatives displayed antioxidant activity greater than that of fenoterol.

WB1106, a novel nitric oxide-releasing derivative of telmisartan, inhibits hypertension and improves glucose metabolism in rats

Angiotensin converting enzyme (ACE) inhibitors usually cause severe coughing and intolerance while antagonists for angiotensin AT 1 receptor do not stimulate the production of nitric oxide (NO). NO has been shown to regulate arterial hypertension and insulin resistance. Hence, new hybrids of antagonist for angiotensin AT 1 receptor and a NO donor may have potent anti-hypertensive effect and regulate glucose metabolism and insulin resistance. Herein, the effects of [6-(nitrooxymethyl)pyridin-2-yl] methyl 4′-[1-(1,7′-dimethyl-2′-propyl-1 H,3′ H-2,5′-bibenzo[ d]imidazol-3′-yl)ethyl] biphenyl-2-carboxylate (WB1106), a novel NO-releasing derivative of telmisartan newly synthesized, on the vasocontraction, hypertension and diet-induced insulin resistance were examined in vitro using rat aortic strips and in normotensive and spontaneous hypertension rats (SHR rats). Apparently, WB1106 induced the vasorelaxation of contracted rat aortic strips in a dose- and time-dependent manner, which depended on the activity of guanylate cyclase, a characteristic of NO-related function. Furthermore, WB1106 reduced the contractile and blood pressure responses to angiotensin II, which relied on the release of telmisartan. Moreover, treatment with WB1106 significantly reduced the blood pressure with similar potency to telmitarsan and increased the contents of cGMP in SHR rats. Therefore, WB1106 possesses both the angiotensin AT 1 receptor antagonist activity of telmisartan and the NO-releasing property of a ‘slow NO donor’. Importantly, in contrast to equimolar telmisartan, treatment with WB1106 significantly attenuated body weight gains and improved glucose tolerance in high-fat and carbohydrate-fed rats, reflecting a synergistic effect of NO and telmisartan. Potentially, WB1106 may be a potent anti-hypertensive drug for treatment of hypertension and diabetes-related cardiovascular diseases in the clinic.

A new class of analgesic agents toward prostacyclin receptor inhibition: Synthesis, biological studies and QSAR analysis of 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazolines

By studying the structural similarity of analgesic imidazolines and 2-phenylnitronyl nitroxides, 20 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazolines ( 2a– t) were newly synthesized as selective antagonists of prostacyclin receptor (IP receptor). In the in vivo tail-flick assay, 2a– t (dose, 0.13 mmol/kg) receiving mice showed increased pain thresholds ranging from 20.52 ± 7.25% to 90.94 ± 11.97%, which were significantly higher than that ranged from 12.27 ± 9.56% to 17.71 ± 7.00% shown by normal saline (NS) receiving mice. In the in vivo tail bleeding assay, 2a– t (dose, 1.30 mmol/kg) receiving mice gave a bleeding time ranging from 116.3 ± 8.0 s to 119.6 ± 7.1 s, and NS receiving mice gave a bleeding time ranging from 116.7 ± 7.5 s to 119.1 ± 8.7 s, which were at a substantially equal level. These observations imply that no bleeding risk occurred even when 10-fold dose of analgesic assay was used. In the in vitro vasorelaxation assay, it was found that when the aortic strip contracted by noradrenaline (NE, final concentration, 10 −7 M) was treated with the solution of 2a– t in NS (final concentration, 5 × 10 −3 M) only lower percentage inhibitions ranged from 6.63 ± 2.72% to 46.28 ± 2.63% were recorded. Relatively higher concentration of 2a– t (5 × 10 −3 M) and relatively lower percentage inhibitions (13 of 20 less than 23.27 ± 3.47%) suggest that 2a– t exhibit few vasodilation activity. To shed some light on the potential analgesic mechanisms of 2a– t, moreover, a QSAR analysis was carried out by using the multiple linear regression method. Taken altogether, the current study confirms that as selective antagonist of IP receptor 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazoline may be a promising lead compound of analgesic agent without cardiovascular and bleeding side effects.

Expression, purification and characterization of human urodilatin in E. coli

Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx–urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02 ± 0.36) × 10 −6 mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5 mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.

NO‐donor melatonin derivatives: synthesis and in vitro pharmacological characterization

Numerous studies document that melatonin possesses a broad-spectrum antioxidant activity. It traps a number of reactive oxygen species (ROS) such as hydroxyl and peroxyl radicals, singlet oxygen and hypochlorous acid. It also inhibits peroxynitrite-induced reactions. It is known that atherosclerosis progression involves ROS-induced oxidation of low-density lipoproteins in sub-endothelial space and the depletion of nitric oxide (NO) in blood vessels, as well as a decreased sensitivity of the vessels to the actions of NO. Considering this, a series of new NO-donor antioxidants were designed and synthesized by joining melatonin with NO-donor nitrooxy and furoxan moieties as polyvalent agents potentially useful for the treatment of cardiovascular diseases involving atherosclerotic vascular changes. The in vitro antioxidant properties of the resulting products were assessed in the thiobarbituric acid reactive substances assay (TBARS), the ABTS(+.) as well as in the alkaline phosphatase (ALP) assay. The antioxidant capacities of NO-donor melatonins to inhibit lipoperoxidation (TBARS-IC(50)) was predominantly dependent on their lipophilicity, and therefore on their partitioning process into membranes. On the other hand, their comparable capacity to inhibit protein oxidation (ALP-IC(50)) was independent of their lipophilicity and was consistent with their similar ability to participate in electron transfer reactions. All the NO-donor melatonins were also evaluated for their ability to relax rat aorta strips precontracted with 1 microM phenylephrine. Finally, binding affinities and intrinsic activity studies, carried out at MT(1) and MT(2) receptor subtypes, showed a rather complex picture in need of further investigation.

NO-Donor COX-2 Inhibitors. New Nitrooxy-Substituted 1,5-Diarylimidazoles Endowed with COX-2 Inhibitory and Vasodilator Properties

A series of NO-donor diarylimidazoles derived from the lead compound Cimicoxib were synthesized and evaluated for their COX-2 inhibitory activity and their stability in whole blood as well as for vasodilator properties. The products are partly transformed into the corresponding alcohols following 24-h incubation in whole blood. All of them display good COX-1/COX-2 selectivity, but are less potent than the lead; a molecular modeling study was carried out to investigate their binding mode. The compounds are also capable of relaxing rat aorta strips precontracted with phenylephrine with a NO-dependent mechanism; this property could confer reduced cardiotoxicity with respect to traditional COX-2 inhibitors.

Novel 1-oxyl-2-substitutedphenyl-4,4,5,5-tetramethylimidazolines: Synthesis, selectively analgesic action, and QSAR analysis

Based on the knowledge that imidazoline can result in analgesic action due to its selective binding with the prostacyclin receptor, 20 1-oxyl-2-substitutedphenyl-4,4,5,5-tetramethylimidazolines ( 3a– t) were prepared in moderate yields. At 0.13 mmol/kg dose, their in vivo analgesic activities were evaluated after the mice were administered at 30, 60, 90, and 150 min. Compared with the pain threshold (12.27 ± 9.56–17.71 ± 7.00%) of normal saline (NS) receiving mice, the pain threshold (23.42 ± 8.14% to 102.58 ± 10.66%) of 3a– t receiving mice increases significantly. Considering a prostacyclin receptor targeting analgesic agent usually had bleeding action and to appraise the bleeding risk, the in vivo tail bleeding time of 1.30 mmol/kg 3a– t receiving mice was found to be ranged from 116.3 ± 8.2 s to 120.3 ± 9.2 s, which was substantially equal to that (117.8 ± 8.4 s to 119.0 ± 8.6 s) of NS receiving mice. Based on the possibility of imidazoline acting as vasodilator, the in vitro vasorelaxations of 3a– t were tested using the rat aortic strip model. When the aortic strip contracted by noradrenaline (NE, final concentration 10 −7 mol/l) was treated with 3a– t (final concentration 5 × 10 −4 mol/l), only lower percentage inhibitions (6.55 ± 5.70–37.40 ± 4.07%) were recorded, implying that the vasorelaxation of 3a– t was neglectable. By selecting appropriate molecular descriptors generated from e-dragon server, the QSAR model of the analgesic activities of 3a– t was constructed using the multiple linear regression method. The established QSAR model showed reasonable accuracy and thus it is promising to be used for screening new 1-oxyl-2-substitutedphenyl-4,4,5,5-tetramethylimidazoline derivatives as analgesic agents.