Rat Caudal Artery Research Articles

Silk fibroin-based embolic agent for transhepatic artery embolization with multiple therapeutic potentials

BackgroundThe excellent physicochemical and biomedical properties make silk fibroin (SF) suitable for the development of biomedical materials. In this research, the silk fibroin microspheres (SFMS) were customized in two size ranges, and then carried gold nanoparticles or doxorubicin to evaluate the performance of drug loading and releasing. Embolization efficiency was evaluated in rat caudal artery and rabbit auricular artery, and the in vivo distribution of iodinated SFMS (125I/131I-SFMS) after embolization of rat hepatic artery was dynamically recorded by SPECT. Transhepatic arterial radioembolization (TARE) with 131I-SFMS was performed on rat models with liver cancer. The whole procedure of selective internal radiation was recorded with SPECT/CT, and the therapeutic effects were evaluated with 18 F-FDG PET/CT. Lastly, the enzymatic degradation was recorded and followed with the evaluation of particle size on clearance of sub-micron silk fibroin.ResultsSFMS were of smooth surface and regular shape with pervasive pores on the surface and inside the microspheres, and of suitable size range for TAE. Drug-loading functionalized SFMS with chemotherapy or radio-sensitization, and the enhanced therapeutic effects were proved in treating HUH-7 cells as lasting doxorubicin release or more lethal radiation. For artery embolization, SFMS effectively blocked the blood supply; when 131I-SFMS serving as the embolic agent, the good labeling stability and embolization performance guaranteed the favorable therapeutic effects in treating in situ liver tumor. At the 5th day post TARE with 37 MBq/3 mg 131I-SFMS per mice, tumor activity was quickly inhibited to a comparable glucose metabolism level with surrounding normal liver. More importantly, for the fragments of biodegradable SFMS, smaller sized SF (< 800 nm) metabolized in gastrointestinal tract and excreted by the urinary system, while SF (> 800 nm) entered the liver within 72 h for further metabolism.ConclusionThe feasibility of SFMS as degradable TARE agent for liver cancer was primarily proved as providing multiple therapeutic potentials.Graphical abstract

Cyclic AMP but Not Calmodulin as a Potential Wasoconstrictor in Simulated Reperfusion.

The phenomena of ischemia and reperfusion are associated with the pathological background of cardiovascular diseases. Ischemia is initiated by ischemia reperfusion injury (IRI), which involves disruption of intracellular signaling pathways and causes cell death. The aim of this study was to assess the reactivity of vascular smooth muscle cells in the conditions of induced ischemia and reperfusion, and to determine the mechanisms leading to contractility disorders. This study was conducted using classical pharmacometric methods on an isolated model of the rat caudal artery. The experiment consisted of the analysis of the final and initial perfusate pressure measurements after induction of arterial contraction with phenylephrine in the presence of forskolin and A7 hydrochloride, two ligands modifying the contractility of vascular smooth muscle cells (VSMC). The pharmacometric analysis showed that in simulated reperfusion, cyclic nucleotides have a vasoconstrictive effect, and calmodulin has a vasodilating effect. The responsiveness of vascular smooth muscle cells to the vasopressor effects of α1-adrenomimetics during reperfusion may change uncontrollably, and the effects of secondary messengers may be counter physiological. Further studies are needed to evaluate the function of other second messengers on VSMCs in the process of ischemia and reperfusion.

Nitrobenzoxadiazole derivatives of the rat selective toxicant norbormide as fluorescent probes for live cell imaging

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats, but relatively harmless to other rodents and mammals. As a vasoactive agent, NRB induces a species-specific vasocontractile effect that is restricted to the peripheral arteries of the rat. Despite the precise mechanisms behind this phenomenon having yet to be fully clarified, it is postulated that the molecular target of NRB could be located within the plasma membrane of rat peripheral artery myocytes (e.g. rat caudal artery myocytes). As such, the primary objective of this study was to develop a fluorescently labelled derivative of NRB to investigate its subcellular distribution/localization in both NRB-sensitive (freshly isolated rat caudal artery myocytes, FIRCAMs) and NRB-insensitive (human hepatic stellate, LX2) cells. Of the examples prepared, lead structure endo-NRB-NBD-bPA subsequently demonstrated retention of the parent toxicant’s pharmacological profile (in terms of its ability to induce both a vasocontractile response in rat caudal artery rings in vitro, and a lethal end-point in rats in vivo). Endo-NRB-NBD-bPA was also shown to be significantly less permeable (an integral feature in the design of fluorescent probes targeting cell-surface receptors) to both LX2 cells and FIRCAMs. Disappointingly, no fluorescence could be observed on the plasma membrane of FIRCAMs stained with endo-NRB-NBD-bPA.

Angiotensin (ang) 1-7 inhibits ang II-induced atrial fibrosis through regulating the interaction of proto-oncogene tyrosine-protein kinase Src (c-Src) and Src homology region 2 domain-containing phosphatase-1 (SHP-1))

ABSTRACT To verify whether Ang-(1-7) produces an antagonistic effect on Ang II-mediated atrial remodeling. Ang II–induced HL-1 cell model and a rat model of Ang II–induced atrial remodeling were constructed and intervened with Ang II Ang-(1-7), AngII +Ang-(1-7), Ang II+ c-Src specific inhibitor (SU6656), and Ang II + Ang-(1-7) + SSG (SHP-1/2 specific inhibitor, stibogluconate), respectively. The systolic blood pressure of the rat caudal artery was detected. And trial fibrosis was detected by Picrosirius red staining and Masson’s trichrome staining. Expressions of transforming growth factor-β (TGF-β), tissue inhibitor of metalloproteinases 1 (TIMP1), Matrix metalloproteinase 2 (MMP-2), connective tissue growth factor (CTGF), galectin-3, α-smooth muscle actin (α-SMA), and collagen I/III were subjected to qPCR and western blot. Furthermore, SHP-1 binding to c-Src was verified by co-immunoprecipitation (Co-IP). Results showed that the expressions of TGF-β, TIMP1, MMP-2, CTGF, α-SMA, galectin-3, and collagen I were increased markedly in the Ang II intervention group, and the expressions of p-ERK1/2, p-Akt, and p-p38MAPK were also increased dramatically. Ang-(1-7) or SU6656 addition could inhibit the action of Ang II factor, thereby minimizing the expressions of the previously described genes and proteins. Simultaneously, SSG supplement reversed the antagonistic effect of Ang-(1-7) on Ang II, and the latter elevated the blood pressure and induced atrial fibrosis in rats. Ang-(1-7) could reverse the changes related to Ang II–induced atrial fibrosis in rats. In conclusion, Ang-(1-7) antagonized Ang II–induced atrial remodeling by regulating SHP-1 and c-Src, thereby affecting the MAPKs/Akt signaling pathway.

Distal Arterialized Venous Supercharging Improves Perfusion and Survival in an Extended Dorsal Three-Perforasome Perforator Flap Rat Model.

Perforator flaps are commonly applied for a variety of skin defects. Many strategies (e.g., hyperbaric oxygen and preconditioning) have been investigated to improve flap survival, but a postoperative 2.03 to 18.2 percent flap necrosis frequency remains a major complication. The authors hypothesized that a distal arterialized venous supercharged (DAVS) flap procedure might improve perfusion and survival in an extended three-perforasome perforator flap rat model and rescue flap ischemia intraoperatively. One hundred twenty male Sprague-Dawley rats (200 to 300 g) were divided into the thoracodorsal artery (TDA) flap group and the DAVS flap group (n = 60 per group). An approximately 11 × 2.5-cm2 flap based on the TDA perforasome was designed in the TDA flap. A DAVS flap was designed based on the TDA flap and supercharged by anastomosing the rat caudal artery with the deep circumflex iliac vein. At postoperative times 1, 3, 6, and 12 hours and 1, 3, 5, and 7 days, perfusion and angiography were compared. On day 7, flap viability and angiogenesis were assessed using histology and Western blotting. The DAVS flap showed a higher survival rate compared with the TDA flap (100 percent versus 81.93 ± 5.38 percent; p < 0.001). All blood flow ratios of deep circumflex iliac artery to TDA perforasome and of choke zone II to choke zone I were higher in the DAVS flap (all p < 0.05). Angiography qualitatively revealed that choke vessels in choke zone II dilated earlier and extensively in the DAVS flap group. CD34+ vessels (68.66 ± 12.53/mm2 versus 36.82 ± 8.99/mm2; p < 0.001) and vascular endothelial growth factor protein level (0.22 ± 0.03 versus 0.11 ± 0.03; p < 0.001) were significantly increased in the DAVS flap group. The DAVS procedure improves three-perforasome perforator flap survival and can be used for rescuing flap ischemia intraoperatively. Further study is needed before possible clinical adoption for reconstructive operations.

The Selective Rat Toxicant Norbormide Blocks KATP Channels in Smooth Muscle Cells But Not in Insulin-Secreting Cells.

Norbormide is a toxicant selective for rats to which it induces a widespread vasoconstriction. In a recent paper, we hypothesized a role of ATP-sensitive potassium (KATP) channels in norbormide-induced vasoconstriction. The current study was undertaken to verify this hypothesis by comparing the effects of norbormide with those of glibenclamide, a known KATP channel blocker. The whole-cell patch-clamp method was used to record KATP currents in myocytes freshly isolated from the rat and mouse caudal artery and from the rat gastric fundus, as well as in insulin-secreting pancreatic beta cells (INS-1 cells). Smooth muscle contractile function was assessed on either rat caudal artery rings or gastric fundus strips. Molecular modeling and docking simulation to KATP channel proteins were investigated in silico. Both norbormide (a racemic mixture of endo and exo isomers) and glibenclamide inhibited KATP currents in rat and mouse caudal artery myocytes, as well as in gastric fundus smooth muscle cells. In rat INS-1 cells, only glibenclamide blocked KATP channels, whereas norbormide was ineffective. The inhibitory effect of norbormide in rat caudal artery myocytes was not stereo-specific as both the endo isomers (active as vasoconstrictor) and the exo isomers (inactive as vasoconstrictor) had similar inhibitory activity. In rat caudal artery rings, norbormide-induced contraction was partially reverted by the KATP channel opener pinacidil. Computational approaches indicated the SUR subunit of KATP channels as the binding site for norbormide. KATP channel inhibition may play a role in norbormide-induced vasoconstriction, but does not explain the species selectivity, tissue selectivity, and stereoselectivity of its constricting activity. The lack of effect in INS-1 cells suggests a potential selectivity of norbormide for smooth muscle KATP channels.

Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging.

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12.Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively.Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division.Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells.

Effects of small interfering RNA targeting TLR4 on expressions of adipocytokines in obstructive sleep apnea hyponea syndrome with hypertension in a rat model.

We explored the effects of RNA interference-mediated silencing of TLR4 gene on expressions of adipocytokines in obstructive sleep apnea hyponea syndrome (OSAS) with hypertension in a rat model. Systolic blood pressure of caudal artery and physiological changes were observed when establishing rat models of OSAS with hypertension. Mature rat adipocytes were induced from separated and cultured primary rat adipocytes. To transfect rat mature adipocytes, TLR4 siRNA group and negative control (NC) siRNA group were established. Expressions of TLR4 mRNA of adipocytes were examined after silenced by siRNA by quantitative real-time polymerase chain reaction (qRT-PCR). By enzyme-linked immunosorbent assay (ELISA), expressions of inflammatory cytokines, and adipocytokines of adipocytes were detected. Blood pressure in rat caudal artery was higher in the intermittent hypoxia group than that of the blank control group by 29.87 mmHg, and cardiocytes in the former group showed physiological changes, which indicated successful establishment of rat models of OSAS with hypertension. Red particles could be seen in mature rat adipocytes when stained with Oil Red O. Transfection of TLR4 mRNA was significantly suppressed in the TLR4 siRNA group, which didn't happen in the untransfected control group. Rats in the TLR4 siRNA group had significantly reduced expressions of such inflammatory cytokines as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) and such adipocytokines as visfatin, adiponectin (ADN), and leptin than those in the untransfected control group. RNA interference-mediated silencing of TLR4 gene could regulate occurrence and development of OSAS with hypertension in rats by downregulating expressions of adipocytokines.

Changes in Sympathetic Innervation of Rat Caudal Artery in Experimental Myocardial Infarction. Effect of Semax Peptide.

Activation of the sympathetic nervous system aggravates the course of myocardial infarction. Semax peptide moderated the degree of this activation and prevented the increase in the density of sympathetic endings in rat caudal artery in 28 days after ischemia or ischemia/reperfusion. The peptide reduced the density of α-adrenoreceptors in the caudal artery of rats with myocardial infarction. Semax produced no effect on β-adrenoreceptors in both experimental models. The experiments on isolated segments of the caudal artery revealed reduced vascular responsiveness to electrical stimulation and norepinephrine infusion in rats treated with Semax after ischemia/reperfusion injury.

Fatty Acid-Derived Pro-Toxicants of the Rat Selective Toxicant Norbormide.

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, as an acute vasoactive, NRB has a rapid onset of action which makes it relatively unpalatable to rats, often leading to sublethal uptake and accompanying bait shyness. A series of NRB-derived pro-toxicants (3a-i, 4a-i, and 5a-i) were prepared in an effort to 'mask' this acute response and improve both palatability and efficacy. Their synthesis, invitro biological evaluation (vasocontractile response in rat vasculature, stability in selected rat media) and palatability/efficacy in Sprague-Dawley, wild Norway, and wild ship rats is described. Most notably, pro-toxicant 3d was revealed to be free of all pre-cleavage vasoconstrictory activity in rat caudal artery and was subsequently demonstrated to release NRB in the presence of rat blood, liver, and pancreatic enzymes. Moreover, it consistently displayed a high level of acceptance by rats in a two-choice bait-palatability and efficacy trial, with accompanying high mortality. On this evidence, fatty acid ester prodrugs would appear to offer a promising platform for the further development of NRB-derived toxicants with enhanced palatability and efficacy profiles.

PM373 In Vivo Assessment of Biodistribution and Efficacy of Bone Marrow Mononuclear Cells for Ischaemia-mediated Neovascularisation

Withdrawn GHEART Vol 9/1S/2014 j March, 2014 j POSTER/2014 WCC Posters PM375 Endothelin-1 Differentially Activates Protein Kinase C and Rho Kinase in Large and Small Arteries Kanchani Rajopadhyaya, Jessica Maddern, John Beltrame, David Wilson, Amenah Jaghoori* Medicine, The University of Adelaide, Vascular Diseases and Therapeutics Reserach Group, The Basil Hetzel Institute For Medical Research, Physiology, The University of Adelaide, Cardiology, The Queen Elizabeth Hospital, Adelaide, Australia Introduction: The clinical management of vasospastic disorders is focused on the blockade of extracellular Ca entry and or specific G-protein coupled receptors. However these therapies neglect numerous additional modes of Ca entry and often do not resolve small vessel dysfunction. This warrants investigating whether there are fundamental differences in the ways that large and small arteries constrict to a given agonist. Objectives: This study aimed to identify whether protein kinase C (PKC) and Rho kinase (ROK)-mediated Ca sensitisation is differentially regulated in large vs small vessels in an acute endothelin-1 (ET-1) model. Methods: Using wire myography, the vascular response to ET-1 was determined acutely in rat caudal arteries following incubation with pharmacological inhibitors. Results: In large caudal arteries (500mm), PKC inhibition prior to ET-1 attenuated the sustained phase of ET-1-mediated vasoconstriction (p 0.05) but did attenuate constriction in the large caudal artery (p<0.05). Conclusion: Differential regulation of PKC and ROK in small and large arteries emphasises the value in considering independent management of large and small vascular disorders. These data indicate that prophylactic ROK inhibition may be an effective treatment strategy for large vessel hyper-reactivity while combined PKC/ROK inhibition, but not ROK inhibition alone may be useful in the treatment of small vessel vasoconstriction. Disclosure of Interest: None Declared

(-)Doxazosin is a necessary component for the hypotensive effect of (±)doxazosin during long-term administration in conscious rats.

Doxazosin is a racemic mixture of (-)doxazosin and (+)doxazosin that is currently used as an add-on therapy for hypertension. In this study we investigated the contribution of each enantiomer to the hypotensive action of long-term administration of (±)doxazosin in conscious rats. Blood pressure of conscious SD rats was measured using a volume pressure recording system. The rats were orally administered (-)doxazosin, (+)doxazosin, or (±)doxazosin (8 mg·kg(-1)·d(-1)) for 12 weeks. Plasma concentrations of the agents were analyzed with HPLC. The effect of the agents on α1-adrenoceptor was examined in isolated rat caudal artery preparations. Treatment of conscious rats with a single dose of (±)doxazosin (8 mg/kg) did not affected DBP and MBP, but significantly decreased SBP by 11.9% 4 h after the administration. Long-term treatment of conscious rats with (±)doxazosin significantly decreased SBP, DBP and MBP with a maximal decrease of SBP by 29.3% 8 h after the last administration. The rank order of the hypotensive actions caused by long-term treatment in conscious rats was (±)doxazosin>(+)doxazosin>>(-)doxazosin. However, the pKB values for inhibiting NA-induced contraction of isolated rat caudal artery were (+)doxazosin (8.995)>(±)doxazosin (8.694)>(-)doxazosin (8.032). The plasma concentrations of (-)doxazosin, (+)doxazosin, and (±)doxazosin were 18.26±3.55, 177.11±20.66, and 113.18±13.21 ng/mL, respectively, 8 h after the last administration of these agents. Long-term treatment with (±)doxazosin produces potent hypotensive action in conscious rats that seems to result from synergic interaction of the two enantiomers.

Effect of Perfluoran on the Morphology of a Red Blood Cell in Acute Blood Loss

Objective: to evaluate the cytoprotective effect of perfluoran when pre-administered in massive blood loss. Material and methods. Experiments were performed on 21 nembutal-anesthetized rats. Acute blood loss from the rat caudal artery served as a model for a terminal state. Perfluoran had been injected 45 minutes prior to blood loss. Morphological and morphome-tric analyses of monolayer dried unstained blood smear slides were made using an Olympus BX-50 light microscope at a magnification of 1000 (10X100). Cytometry was carried out using Image scope color software. Results. In the control group of experiments, i. e. without perfluoran preadministration, at 5 minutes of blood loss, the circulating blood showed only 20% of discoid forms; the other forms of the red blood cells were grades 1-to-3 transformation stomatocytes. By 60 minutes of hypotension, there was a decrease in stomatocyte counts to 5.2% and a change in the ratio of circulating red blood cell populations. The number of smaller, i.e. senescent, cells was increased and that of large cells was reduced. The preventive administration of perfluoran in small amounts playing no prominent role in the delivery of oxygen and the alteration of circulating blood volume, substantially changed the ratio of different shapes and sizes of erythrocytes, increased their average diameter, and stabilized the membrane of a red blood cell. Conclusion. The preadministration of low-dose perfluoran before blood loss prevents deformational stress, increases the surface of a red blood cell, and stabilizes its membrane. Key words: blood loss, perfluoran, red blood cell, stomatocyte, deformation stress, rheology.

Effects of Estrogen on Relaxation Rates in Rat Caudal Artery

Menopausal women are more at risk for heart problems such as hypertension, which could arise from altered arterial smooth muscle function. If reproductive hormones impact cardiovascular health by altering vascular muscle contractility, we should note changes in the arteriolar contractility as cyclic hormone levels change. This project studies the effect of varying reproductive hormone levels on relaxation rates in rat caudal arteries. Retired breeding rats experience a peak and decline in natural estrogen and progesterone levels every four days. Experiments were performed on rat caudal artery collected during various phases of the reproductive cycle. Vaginal epithelial cell cytology indicated hormone levels. Isometric tension was recorded after electrical field stimulation of vascular tissue in a muscle bath under physiological conditions. Relaxation curves analyzed using JSIM modeling software matched a double exponential decay curve. The changing hormone levels caused no difference in the relaxation rates for either the slow or the fast portion of the relaxation curve. These results imply that the protective effect of natural hormone replacement therapy may not be directly impacting vascular function as a preventative measure for a healthy heart.

The Effects of Estrogen and Progesterone on Relaxation Rates in Rat Caudal Artery

The Effects of Estrogen and Progesterone on Relaxation Rates in Rat Caudal Artery

Prostate-apoptosis response-4 phosphorylation in vascular smooth muscle

The protein prostate-apoptosis response (Par)-4 has been implicated in the regulation of smooth muscle contraction, based largely on studies with the A7r5 cell line. A mechanism has been proposed whereby Par-4 binding to MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase, MLCP) blocks access of zipper-interacting protein kinase (ZIPK) to Thr697 and Thr855 of MYPT1, whose phosphorylation is associated with MLCP inhibition. Phosphorylation of Par-4 at Thr155 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition and contraction. We tested this “padlock” hypothesis in a well-characterized vascular smooth muscle system, the rat caudal artery. Par-4 was retained in Triton-skinned tissue, suggesting a tight association with the contractile machinery, and indeed Par-4 co-immunoprecipitated with MYPT1. Treatment of Triton-skinned tissue with the phosphatase inhibitor microcystin (MC) evoked phosphorylation of Par-4 at Thr155, but did not induce its dissociation from the contractile machinery. Furthermore, analysis of the time courses of MC-induced phosphorylation of MYPT1 and Par-4 revealed that MYPT1 phosphorylation at Thr697 or Thr855 preceded Par-4 phosphorylation. Par-4 phosphorylation was inhibited by the non-selective kinase inhibitor staurosporine, but not by inhibitors of ZIPK, Rho-associated kinase or protein kinase C. In addition, Par-4 phosphorylation did not occur upon addition of constitutively-active ZIPK to skinned tissue. We conclude that phosphorylation of Par-4 does not regulate contraction of this vascular smooth muscle tissue by inducing dissociation of Par-4 from MYPT1 to allow phosphorylation of MYPT1 and inhibition of MLCP.

Prostate Cell Membrane Chromatography–Liquid Chromatography–Mass Spectrometry for Screening of Active Constituents from Uncaria rhynchophylla

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography-mass spectrometry (LC-MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC-MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors.

Cross-talk between Rho-associated Kinase and Cyclic Nucleotide-dependent Kinase Signaling Pathways in the Regulation of Smooth Muscle Myosin Light Chain Phosphatase

Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.

Effect of tissue engineering small vessel scaffolds in experimental animals

Acellular xenogeneic small vessel scaffolds preliminarily eliminate the xenogeneic antigen that could induce an immune rejection. We transplanted the Wistar rat caudal artery scaffolds into Japanese white rabbit ear central arteries, and observed the changes of blood flow and vessel wall. We acquired 30 caudal arteries from 15 Wistar rats, some were directly assigned as caudal artery group, the others were disposed by 1% p-octyl polyethelene glycol phenyl ether, and were assigned as the group of tissue engineered small vessel scaffold. We collected 15 Japanese white rabbits, and utilized a pair of ear central arteries per rabbit as hosts. Through microscopic surgery, the proximal broken end of central artery was overlapped into the donor’s proximal vessel lumen by sleeve anastomosis, while the distal broken end was sutured by classical end-to-end anastomosis. The maximum patency time of small vessel scaffold was 46 hours and 47 minutes, and which of caudal vessel was 14 hours. The poor blood flow primarily was occurred in distal broken end, while proximal end was connected in pedicle graft structure. On the 10th day, tunica intima fibers in vessel scaffold were still arranged in cylindrical conformation regularly without cell adherence. On the 100th day, tunica intima fibers were still preserved completely in proximal end. The results showed that xenogeneic acellular small vessel scaffold can be transplanted into the host, and the sleeve anastomosis was significantly superior to the classical end-to-end anastomosis.

Age-dependent vasorelaxation of Casimiroa edulis and Casimiroa pubescens extracts in rat caudal artery in vitro

Leaf and seed decoctions of Casimiroa spp. are used in Mexican traditional medicine to treat high blood pressure. Previous researches showed as Casimiroa extracts are able to induce relaxation of rat aortic and caudal arteries. To study the influence of the aging, we determined the vascular effect induced by extracts of Casimiroa edulis and Casimiroa pubescens in arterial tissues from young and old rats. The activity of Casimiroa edulis extracts: hexanic-leaf (Ce5), methanolic-leaf (Ce6), hexanic-seed (Ce7) and methanolic-seed (Ce8), and Casimiroa pubescens: hexanic-leaf (Cp9), methanolic-leaf (Cp10), hexanic-seed (Cp11) and methanolic-seed (Cp12) were investigated in precontracted caudal arteries of young (4 months) and old (20 months) rats. The Casimiroa extracts tested at 20 μg/ml induced vasorelaxation in phenylephrine-precontracted arterial tissues both in young and old arterial tissues. Methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12) caused a higher relaxation in young than in old arterial tissues. Nifedipine (0.01 μM) did not change the vasorelaxation induced by Casimiroa edulis extract either in young and old rat arterial tissues. The vasorelaxation induced by Casimiroa edulis and Casimiroa pubescens extracts is decreased from aging since the effects were higher in young than in old rat arterial tissues. However, the methanolic-seed extracts of both plant species induced a relevant vasorelaxation also in old arterial tissues. Thus the results support the traditional use of Casimiroa decoctions as antihypertensive, also in elderly.