Live Imaging and Analysis of Vasoactive Properties of Drugs Using an in-ovo Chicken Embryo Model: Replacing and Reducing Animal Testing.

Vasodilation occurs as a result of the relaxation of the smooth muscle cells present in the walls of blood vessels. Various suitable models are available for the analysis of the vasoactive properties of drugs with therapeutic applications. But all these models have limitations, such as ethical issues and high cost. The purpose of this study is to develop an alternative model for studying the vasoactive properties of drugs using an in-ovo chicken embryo model. In the preliminary experiment, we used a well-known vasoconstrictor (adrenaline) and a vasodilator (spermine NoNoate) in the chick embryo area vasculosa and evaluated their concentration-response curve. Adrenaline (10 µM) and spermine NoNoate (10 µM) were administered in different arteries and veins and different positions of the right vitelline artery of the chick embryo. Results showed the middle of the vessel bed of the right vitelline artery having the best vasoactive effect compared to others. Finally, anti-hypertensive drugs, calcium channel blockers, and NOS agonists were administered in the chick embryo area vasculosa to validate the model. Results demonstrate that the chick embryo area vasculosa can be an alternative, robust, and unique in-ovo model for screening of anti-hypertensive drugs in real time.

Isoprenyl phenolic ethers from the termite nest-derived medicinal fungus Xylaria fimbriata.

Seven new isoprenyl phenolic ethers, namely fimbriethers A‒G (1‒7), were isolated from the fermented broth of the termite nest-derived medicinal fungus Xylaria fimbriata YMJ491. Their structures were determined by spectroscopic data analysis and compared with those reported. The effects of all the isolates at a concentration of 100 µM on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells were evaluated, and all of them exhibited NO production inhibitory activity with Emax values ranging from 4.6 ± 2.0% to 49.7 ± 0.5% without significant cytotoxicity. In addition, these seven compounds did not alter phenylephrine-induced vasocontraction in isolated intact thoracic aortic rings from C57BL/6J mouse, indicating 1‒7 were not involved in the regulation of endothelial NOS-mediated NO production.

A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting.

Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives.

Development and Validation of Stability-Indicating HPTLC Method for Estimation of Naratriptan Hydrochloride in Its Pharmaceutical Dosage Form and Its Content Uniformity Testing.

The present research project involves development and validation of a stability-indicating HPTLC method for the estimation of naratriptan-HCl in their pharmaceutical dosage forms and its content uniformity testing. Naratriptan-HCl was subjected to alkaline, acidic, oxidative, neutral, thermal (dry heat) and photo-degradation conditions. The chromatographic separation was carried out using a precoated silica gel G 60 F254 TLC plate as the stationary phase and dichloromethane-toluene-methanol-triethylamine (4 : 4 : 2 : 1, v/v/v/v) as the mobile phase. The spots of NRT-HCl and its degradation products were detected at 290 nm. The Rf value of NRT-HCl was found to be 0.60 ± 0.02. The linearity was obtained in the range of 100-500 ng/spot. The limit of detection and limit of quantitation were found to be 6.07 ng/spot and 18.41 ng/spot, respectively. The percentage recovery was found in the range of 98.87-99.55%. NRT-HCl was degraded under acidic, alkaline and oxidative conditions while stable under photolytic, neutral and dry heat conditions. The developed method was applied for estimation of naratriptan-HCl in marketed formulations and its content uniformity testing.

Cardiovascular effects of Sp-CTx, a cytolysin from the scorpionfish (Scorpaena plumieri) venom.

Fish venom cytolysins are multifunctional proteins that in addition to their cytolytic/hemolytic effects display neurotoxic, cardiotoxic and inflammatory activities, being described as "protein lethal factors". A pore-forming cytolysin called Sp-CTx (Scorpaena plumieriCytolytic Toxin) has been recently purified from the venom of the scorpionfish Scorpaena plumieri. It is a glycoprotein with dimeric constitution, comprising subunits of approximately 65 kDa. Previous studies have revealed that this toxin has a vasorelaxant activity that appears to involve the L-arginine-nitric oxide synthase pathway; however its cardiovascular effects have not been fully comprehended. The present study examined the cardiovascular effects of Sp-CTx in vivo and in vitro. In anesthetized rats Sp-CTx (70 µg/kg i.v) produced a biphasic response which consisted of an initial systolic and diastolic pressure increase followed by a sustained decrease of these parameters and the heart rate. In isolated rats hearts Sp-CTx (10(-9) to 5 × 10(-6) M) produced concentration-dependent and transient ventricular positive inotropic effect and vasoconstriction response on coronary bed. In papillary muscle, Sp-CTx (10(-7) M) also produced an increase in contractile isometric force, which was attenuated by the catecholamine releasing agent tyramine (100 µM) and the ß-adrenergic antagonist propranolol (10 µM). On isolated ventricular cardiomyocytes Sp-CTx (1 nM) increased the L-type Ca(2+) current density. The results show that Sp-CTx induces disorders in the cardiovascular system through increase of sarcolemmal calcium influx, which in turn is partially caused by the release of endogenous noradrenaline.

Vasoconstriction induced by salivary gland extracts from ixodid ticks.

In their quest for blood, most haematophagous parasites secrete vasodilators in their saliva to counter the host haemostatic response of vasoconstriction. Surprisingly, salivary gland extracts from adult female Dermacentor reticulatus and Rhipicephalus appendiculatus ticks induced constriction in a rat femoral artery model; males induced vasoconstriction or vasodilation depending on the time of feeding. Based on comparative HPLC fractionation, the active compounds inducing vasoconstriction do not appear to be prostaglandins (which ticks normally use as vasodilators). Vasoconstriction may be unique to ixodid ticks, helping them control blood flow during their prolonged blood-feeding of up to 10 days or more.

Characterization of two vasoactive peptides isolated from the plasma of the snake Crotalus durissus terrificus.

Incubation of plasma from the snake Crotalus durissus terrificus (CDTP) with trypsin generated two hypotensive peptides. The primary structure of the peptides was established for two sequences as: (Ser-Ile-Pro-Gln-Ala-Pro-Thr-Ser-Asn-Leu-Ile-Glu-Ala-Thr-Lys) and (Lys-Pro-Asp-Ala-Asn-Gln-Val-Leu-Ile-Gln-Val-Ile-Gly-Val). These peptides display homology with fragments of albumin from Trimeresurus flavoviridis. Bolus intra-arterial injection of the purified or the synthetic peptide produced a strong and sustained vasopressor response in the anaesthetized snake (CDT) and rats (Wistar); this hypotensive effect was also potentiated by captopril-an angiotensin-converting (0.1 mg/kg) enzyme inhibitor.

The structure of helokinestatin-5 and its biosynthetic precursor from Gila monster (Heloderma suspectum) venom: evidence for helokinestatin antagonism of bradykinin-induced relaxation of rat tail artery smooth muscle.

Here we report the primary structure of a novel peptide, named helokinestatin-5 (VPPPLQMPLIPR), from the venom of the Gila monster (Heloderma suspectum). Helokinestatin-5 differs in structure from helokinestatin-3 by deletion of a single prolyl residue in the N-terminally located polyproline region. Two different biosynthetic precursors were consistently cloned from a venom-derived cDNA library. The first encoded helokinestatins 1-4 and a single copy of C-type natriuretic peptide, as previously described, whereas the second was virtually identical, lacking only a single prolyl codon as found in the mature attenuated helokinestatin-5 peptide. Helokinestatins 1-3 and 5 were synthesized by solid-phase fmoc chemistry and each synthetic replicate was found to antagonize the relaxation effect induced by bradykinin on rat tail artery smooth muscle. Helokinestatins thus represent a novel family of vasoactive peptides from the venom of helodermatid lizards.

Zooxanthellamide Cs: vasoconstrictive polyhydroxylated macrolides with the largest lactone ring size from a marine dinoflagellate of Symbiodinium sp.

Zooxanthellamide Cs (ZAD-Cs), C(128)H(220)N(2)O(53)S(2) (ca. 2.7 kDa), was obtained from a cultured marine dinoflagellate of the genus Symbiodinium as an inseparable isomeric mixture of polyhydroxylated 61- to 66-membered macrolides. The chemical structures of the components were clarified by detailed 2D NMR analysis to be the macrolactonized analogues of zooxanthellamide A (ZAD-A), which had been previously isolated from the same microalgae. Chemical lability of ZAD-Cs suggests that ZAD-A is an artifact derived from ZAD-Cs during the isolation steps. Three of the components possess the largest (63-, 64-, and 66-membered) ring sizes found to date among the natural macrolides. ZAD-Cs exhibited higher vasoconstrictive activity than that of the zooxanthellatoxins, the first vasoconstrictive macrolides from Symbiodinium sp. The structure-activity relationship suggests that the huge macrolactone structure is important for biological activity. The relationship between the structures of the polyol metabolites and the phylogenetic systematics of Symbiodinium sp. is also discussed.

Cardioactive diterpenoids from the roots of Salvia amplexicaulis.

Five diterpenoids, three steroids, four triterpenoids and one flavonoid were isolated from the roots of Salvia amplexicaulis Lam. (Lamiaceae). Structures of these compounds were elucidated by spectroscopic analysis. The crude extract and the pure compounds were tested for cardiovascular parameters using Wistar Albino rats. The crude extract, and 7-oxo-abieta-9,12,14-triene, ferruginol, stigmast-4-en-3-one showed a vasodepressor effect.

Relaxing and contracting activities of heartworm extract on isolated canine abdominal aorta.

Effect of adult heartworm (HW) crude extract on isolated canine abdominal aortic strips precontracted with noradrenaline was examined by recording isometric changes in tension. HW extract caused contraction of the aortic strip at a low concentration (LC) and its relaxation at a high concentration (HC). In aortic strips without endothelium, LC extract elicited a contraction similar to that in the strips with endothelium, whereas HC extract failed to produce any relaxation but instead produced a contraction. The relaxing effect of HC extract was blocked after treatment with 300 microM NG-nitro-L-arginine methyl ester hydrochloride, with reversal by additional treatment with 3 mM L-arginine. It was also markedly reduced or abolished after treatment with 3 microM oxyhemoglobin or 1 microM methylene blue. Fractionation of HW extract by high-performance liquid chromatography revealed that the relaxing and contracting activities are due to different substances in the extract. The results indicate that HW extract contains 2 different vasoactive substances, 1 causing contraction of canine abdominal aorta via a direct action on the smooth muscle, and the other its relaxation indirectly by releasing nitric oxide from endothelial cells. These vasoactive substances might play a role in HW extract-induced shock in dogs, and in the pathogenesis of HW infection.

[Separation and determination of pseudoephedrine in bufferin cold tablet by capillary electrophoresis with hydropropyl-beta-cyclodextrin as chiral selective reagent].

A method for the separation and analysis of the chiral pseudoephedrine enantiomers using capillary electrophoresis was established. The buffer solution for separation was 25 mmol/L Tris-phosphate, including 38 mmol/L hydropropyl-beta-cyclodextrin with pH value of 2.65. (1S, 2S)(+) Pseudoephedrine in Bufferin Cold tablet was determined. The method has good precision, recovery and linear relationship.

Adrenomedullin (11-26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla.

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11-26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11-26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11-26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.

Isolation and characterization of coenzyme A glutathione disulfide as a parathyroid-derived vasoconstrictive factor.

BACKGROUND: Coenzyme A glutathione disulfide (CoA-SSG) was recently isolated from bovine adrenal glands and was shown to be a renal vasoconstrictor. The identification of CoA-SSG in human parathyroid glands and its action on cultured vascular smooth muscle cells (VSMCs) are described here. METHODS AND RESULTS: After purification to homogeneity by several chromatographic steps, CoA-SSG was identified by matrix-assisted laser desorption/ionization mass spectrometry and enzymatic analysis. The dose-dependent growth-stimulating effect of CoA-SSG on VSMCs, measured by the [(3)H]thymidine method, is characterized by a threshold of 10(-)(8) mol/L and a maximum effect of 10 micromol/L, increasing VSMC proliferation 254+/-21% above control. A dose of 10 micromol/L methylmalonyl-CoA and 10 micromol/L CoA increased the rate of proliferation of VSMCs only by 178+/-43% and 50+/-42% above control, respectively. Glutathione has no proliferative effect on VSMCs. The growth-stimulating effect of CoA-SSG (1 micromol/L) was decreased by the antagonists 3,7-dimethyl-1-propargylxanthine (DMPX; 11 micromol/L) (38% compared with CoA-SSG without antagonist) and pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (PPADS; 10 micromol/L) (48% compared with CoA-SSG without antagonist; each P:<0. 05 versus control), indicating that the effect is mediated partly via A(2) and partly via P(2)Y(1) and/or P(2)Y(4) receptor. CONCLUSIONS: CoA-SSG may play a regulatory role in VSMC growth as a progression factor and thereby could play an important role in development of hypertension.

Peptidergic innervation of the human clitoris.

In the present study, the distributions of neuropeptides in the normal human clitoris and in a clitoris from an adrenogenital syndrome (AGS) was demonstrated by immunohistochemistry (IHC). Immunohistochemical screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), neuropeptide tyrosine (neuropeptide Y), C-flanking peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP) and substance P immunoreactivities. Special attention was given to the VIP-related peptide helospectin, that has been detected in neuronal elements in the clitoris. No visible differences between the localization and distribution of peptidergic nerve fibers of normal and hypertrophic clitoris from AGS have been observed. Co-localization studies showed the co-existence of VIP, PHM and partly helospectin and neuropeptide Y with CPON within nerve fibers in the cavernous tissue and substance P and CGRP co-expression in nerve fibers especially underneath and within the glans clitoris.

Identification and characterization of P(1), P(7)-Di(adenosine-5')-heptaphosphate from human platelets.

Diadenosine pentaphosphate and diadenosine hexaphosphate have been isolated in human platelets and have been postulated to play an important role in the control of vascular tone. Here we describe the isolation and identification of diadenosine heptaphosphate from human platelets. Dinucleoside polyphosphates were concentrated by affinity chromatography from a nucleotide-containing fraction from deproteinated human platelets. Dinucleoside polyphosphates were purified by anion-exchange and reversed phase high performance liquid chromatography to homogeneity. Analysis of one of these fractions with matrix-assisted laser desorption/ionization mass spectrometry revealed a molecular mass of 1076.4 (1077.4 = [M + H](+)) Da. UV spectroscopic analysis of this fraction showed the spectrum of an adenosine derivative. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrum of the fraction minus that of diadenosine heptaphosphate (Ap(7)A) demonstrated that the isolated substance was identical to Ap(7)A. The identity of the retention times of the authentic and the isolated compound confirmed this result. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5'-positions of the riboses. With thrombin-induced platelet aggregation, Ap(7)A is released from the platelets into the extracellular space. The vasoconstrictive action of Ap(7)A on the vasculature of the isolated perfused rat kidney Ap(7)A was slightly less than that of Ap(6)A. The threshold of the vasoconstrictive action of Ap(7)A was 10(-5) mol/liter. The vasoconstrictive effect was abolished by suramin and pyridoxal phosphate 6-azophenyl-2', 4'-disulfonic acid, suggesting an activation of P(2x) receptors. Furthermore, Ap(7)A inhibits ADP-induced platelet aggregation. Thus, the potent vasoconstrictor Ap(7)A derived from human platelets, like other diadenosine polyphosphates, may play a role in the regulation of vascular tone and hemostasis.

Microvascular protective activity of flavonoid glucuronides fraction from Tulipa gesneriana.

A mixture of flavonoid glucuronides, consisting of 7-O-glucuronides of kaempferol and quercetin 3-O-rutinosides, 3-O-gentiobiosides and 3-O-glucosides, was isolated from the perianths of Tulipa gesneriana L. var. 'Paradae'. It showed protective activity against the increased (both chloroform and histamine) skin vascular permeability in rabbits. The protective effect, measured as the reduction in leakage of Evans blue, was 59.8% after peritoneal treatment at a dose of 25 mg/kg, while that of troxerutin was 45.5%.

Adenosine(5') oligophospho-(5') guanosines and guanosine(5') oligophospho-(5') guanosines in human platelets.

We isolated and identified nucleoside(5') oligophospho-(5') nucleosides containing adenosine and guanosine (ApnG; n = 3-6) as well as diguanosine polyphosphates (GpnG; n = 3-6) in human platelets. For identification, UV spectrometry, matrix-assisted laser desorption/ionization, postsource decay matrix-assisted laser desorption/ionization mass spectrometry, and enzymatic cleavage experiments were used. The adenosine(5') oligophospho-(5') guanosines act as vasoconstrictors and growth factors. The diguanosine polyphosphates are potent modulators of growth in vascular smooth muscle cells, but do not affect vascular tone.

Multiple vasoactive factors in epidermal secretions of the Arabian Gulf catfish, Arius bilineatus (Valenciennes).

1. The Arabian Gulf catfish produces proteinaceous epidermal secretions when threatened or injured. 2. The soluble fraction of the catfish epidermal secretions (SES) has vasoconstrictor activities on sheep renal arteries, which can be inhibited by several antagonists, including atropine, indomethacin, prazosin, and verapamil. 3. Mepyramine, yohimbine, and ketanserin have negligible effects on SES-induced contraction. 4. SES exhibits a significant tachyphylaxis upon addition of a second (8.4% reduction) and third (47% reduction) dose of SES to the organ bath, which can be partially prevented by addition of a fresh arterial section prior to each addition. 5. A vasoconstricting activity has been partially purified from SES by gel filtration on Fractogel HW-65(F) and appears to be a protein with a pI near 7.3. This activity is affected only by verapamil and prazosin.

Carbazole alkaloids from Clausena excavata and their biological activity.

Five new carbazole alkaloids, clausines B, E, H, I and K, as well as 22 known compounds, were isolated from the stem bark of Clausena excavata. The structures were established from spectral data and chemical transformation. These compounds showed significant inhibition of rabbit platelet aggregation and caused vasocontraction. The crude methanol extract, partitioned layers and chromatographic fractions revealed the presence of promotive and inhibitive constituents, simultaneously. These results might explain the philosophy of use in Chinese medicine, in that the dose and content variation in a prescription produced different, promotive or inhibitive, effects on therapy.