Felodipine Sensitivity In Vivo and In Vitro of Activation Pathways in Vascular Smooth Muscles

Aspartic acid was replaced in position 1 of angiotensin II (ATII) with several amino acids, to assess the possible influence of the N-terminal amino acid for (a) the intrinsic activity, (b) the affinity, and (c) the metabolic degradation of agonist analogues of ATII. Some of the substitutions in position 1 were used in combination with replacement of Phe by Gly or Leu in position 8, to obtain the corresponding antagonist.The compounds were tested in vivo (rat blood pressure) and in two in vitro preparations (rat stomach and rabbit aorta strips). The oil immersion technique, described by Kalsner and Nickerson (1968) (Can. J. Physiol. Pharmacol. 46, 719–730), was used to study the disposition of the peptides by vascular smooth muscles (rabbit aorta strips). Degradation of the peptides by purified aminopeptidases was evaluated in vitro by measuring the fragments on paper chromatography. Potency of antagonists was estimated in vivo (ID50) and in vitro (pA2 values): duration of action was established by infusing the inhibitors intravenously into anesthetized rats and testing the effect of standard doses of angiotensin before and after.The results indicate that replacement of Asp with other amino acids does not influence the intrinsic activity, but can either increase or decrease the affinity in vitro or the potency in vivo. 1-Sar-ATII, and 1-D-Ala-ATII are more potent and longer acting than 1-Asp-ATII on isolated intestinal and vascular smooth muscles, but not in vivo. On the contrary, 1-β-Asp-ATII and 1-β-D-Asp-ATII are more potent than 1-Asp-ATIIin vivo, but not on rabbit aorta strips. Rate of relaxation of rabbit aorta strips suspended in oil, after contraction with submaximal doses of several analogues of ATII, are significantly slower than relaxation after 1-Asn2-ATII and 1-Asp-ATII. A close parallelism between the diminution of the relaxation rate in oil and the degradation by aminopeptideses in vitro was observed, suggesting that metabolic degradation may be the major factor determining relaxation of rabbit aorta in oil after contraction with one of these peptides. Potencies of antagonists in vivo and in vitro are increased by replacing Asp with Sar. Substitution of Asp with β-Asp or β-D-Asp brings about a slight increase of potency in vivo but not in vitro. It appears that firm binding and prolonged occupation of receptors by sarcosyl derivatives are the primary factors contributing to increase the potency and to prolong the duration of action of antagonists, while prevention or reduction of metabolic breakdown by aminopeptidases is much less efficient.

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Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: Role of microRNA-133a

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00290.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. G964-G973 ◽

Cited By ~ 12

Author(s):

Jagmohan Singh ◽

Ettickan Boopathi ◽

Sankar Addya ◽

Benjamin Phillips ◽

Isidore Rigoutsos ◽

...

Keyword(s):

Smooth Muscle ◽

Anal Sphincter ◽

Internal Anal Sphincter ◽

Smooth Muscles ◽

Smooth Muscle Contractility ◽

Network Analyses ◽

Rhoa Signaling

A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets ( Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A2analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence.

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Mg2+–Ca2+ interaction in contractility of vascular smooth muscle: Mg2+ versus organic calcium channel blockers on myogenic tone and agonist-induced responsiveness of blood vessels

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y87-120 ◽

1987 ◽

Vol 65 (4) ◽

pp. 729-745 ◽

Cited By ~ 174

Author(s):

B. M. Altura ◽

B. T. Altura ◽

A. Carella ◽

A. Gebrewold ◽

T. Murakawa ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Blood Vessels ◽

Divalent Cations ◽

Smooth Muscles ◽

In Vivo Studies ◽

Channel Blockers ◽

Arterial Blood

Contractility of all types of invertebrate and vertebrate muscle is dependent upon the actions and interactions of two divalent cations, viz., calcium (Ca2+) and magnesium (Mg2+) ions. The data presented and reviewed herein contrast the actions of several organic Ca2+ channel blockers with the natural, physiologic (inorganic) Ca2+ antagonist, Mg2+, on microvascular and macrovascular smooth muscles. Both direct in vivo studies on microscopic arteriolar and venular smooth muscles and in vitro studies on different types of blood vessels are presented. It is clear from the studies done so far that of all Ca2+ antagonists examined, only Mg2+ has the capability to inhibit myogenic, basal, and hormonal-induced vascular tone in all types of vascular smooth muscle. Data obtained with verapamil, nimopidine, nitrendipine, and nisoldipine on the microvasculature are suggestive of the probability that a heterogeneity of Ca2+ channels, and of Ca2+ binding sites, exists in different microvascular smooth muscles; although some appear to be voltage operated and others, receptor operated, they are probably heterogeneous in composition from one vascular region to another. Mg2+ appears to act on voltage-, receptor-, and leak-operated membrane channels in vascular smooth muscle. The organic Ca2+ channel blockers do not have this uniform capability; they demonstrate a selectivity when compared with Mg2+. Mg2+ appears to be a special kind of Ca2+ channel antagonist in vascular smooth muscle. At vascular membranes it can (i) block Ca2+ entry and exit, (ii) lower peripheral and cerebral vascular resistance, (iii) relieve cerebral, coronary, and peripheral vasospasm, and (iv) lower arterial blood pressure. At micromolar concentrations (i.e., 10–100 μM), Mg2+ can cause significant vasodilatation of intact arterioles and venules in all regional vasculatures so far examined. Although Mg2+ is three to five orders of magnitude less potent than the organic Ca2+ channel blockers, it possesses unique and potentially useful Ca2+ antagonistic properties.

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Construction and In Vitro Characterization of Attenuated Feline Immunodeficiency Virus Long Terminal Repeat Mutant Viruses

Journal of Virology ◽

10.1128/jvi.75.2.1054-1060.2001 ◽

2001 ◽

Vol 75 (2) ◽

pp. 1054-1060 ◽

Cited By ~ 12

Author(s):

Luisa Bigornia ◽

Kristen M. Lockridge ◽

Ellen E. Sparger

Keyword(s):

Long Terminal Repeat ◽

Virus Replication ◽

Feline Immunodeficiency Virus ◽

Terminal Repeat ◽

Immunodeficiency Virus ◽

Activation Pathways ◽

Virus Expression ◽

Transcriptional Elements

ABSTRACT AP-1- and ATF-binding sites are cis-acting transcriptional elements within the U3 domain of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) that serve as targets for cellular activation pathways and may regulate virus replication. We report that FIV LTR mutant proviruses encoding U3 deletions of the ATF-binding sequence exhibited restricted virus expression and replication in both feline lymphocytes and macrophages. In contrast, deletion of the AP-1 site had negligible effects on virus expression and replication. FIV LTR mutant proviruses encoding deletions of both the AP-1 and ATF sites or a 72-bp deletion encompassing the AP-1 site, duplicated C/EBP sites, and ATF sites were severely restricted for virus expression. These results demonstrate that deletion of either the ATF-binding site or multiplecis-acting transcriptional elements attenuates FIV. These attenuated FIV mutants provide opportunities to characterize the role of cis-acting elements in virus replication in vivo and to test LTR mutants as attenuated virus vaccines.

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Thrombogenicity of Microparticles Derived from Vascular Cells.

Blood ◽

10.1182/blood.v108.11.382.382 ◽

2006 ◽

Vol 108 (11) ◽

pp. 382-382

Author(s):

Hirotaka Isobe ◽

Thomas Perkmann ◽

Olga Oskolkova ◽

Valeri Bochkov ◽

Bernd R. Binder

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Cell Activation ◽

Smooth Muscles ◽

Calcium Ionophore ◽

Vascular Cells ◽

Independent Manner ◽

Thrombin Formation

Abstract Microparticles (MPs) are released from cells during processes such as apoptosis or during cell activation. These MPs contain phospholipids, proteins and even nucleic acids derived from their parent cells. They are found circulating in plasma but also in tissues such as atherosclerotic plaques. It is thought that MPs contain and transfer tissue factor and can thereby induce blood clotting. In this study we analyzed clot promoting properties of MPs generated from vascular cells in vitro. MPs were generated from endothelial cells (EC), smooth muscles cells (SMC), monocytes (U937), erythrocytes (RBC) or platelets (Pl) by inducing apoptosis or by calcium ionophore activation; they were subsequently isolated by differential centrifugation. Thrombogenicity of the MPs was evaluated using a thrombin generation assay (Technothrombin® TGA) and MP free plasma as substrate. MPs displayed a different thrombin generating potential depending on the parent cells. MPs derived from RBCs (~400nM peak thrombin/105 MPs/ml plasma), ECs (~300nM), SMCs (~300nM) and Pls (~300nM) were more thrombogenic than MPs derived from U937 (~200 nM). In addition EC, SMC and U937 MPs all expressed tissue factor but EC MPs induced thrombin generation in a tissue factor and FVII independent manner. EC MPs even expressed active tissue factor pathway inhibitor and functionally inhibited tissue factor dependent thrombin generation. Since the higher thrombin generation induced by MPs derived from EC as compared U937 derived MPs could not be explained by a different activity of tissue factor, we were interested whether lipids contained in the microparticles could account for the differences in thrombin generation. We therefore analyzed thrombin generation induced by lipids isolated from MPs and parent cells and could show that lipids from EC MPs and SMC MPs exhibited higher thrombin generation than those from U937 MPs. Upon analysis of lipids by thin layer chromatography and mass spectrometry we found that in general microparticles are enriched in cholesterol, sphingomyeline and phosphatidylserine over the parent cells and that EC and SMC MPs were enriched in negatively charged phospholipids (different species of phosphatidylserine and phosphatiylglycerol) as compared to MPs derived from U937 cells. When thrombogenicity was, however, evaluated in vivo by injecting MPs into mice it was found that the highest capability to induce thrombin-antithrombin (TAT) complexes had MPs derived from SMCs; also U937 MPs induced an increase in TAT levels, while EC MPs – although more thrombogenic than U937 MPs in vitro – did not induce TAT complex formation by themselves but were only synergistic in vivo. From these data we conclude that thrombin formation in vivo depends on the initiation of the tissue factor FVII pathway, while the extent of thrombin formation is dependent on negatively charged phospholipids contained to a higher extent in MPs derived e.g. from ECs.

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Cardiovascular actions of kinins in the rabbit

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y79-012 ◽

1979 ◽

Vol 57 (1) ◽

pp. 78-91 ◽

Cited By ~ 35

Author(s):

J. Barabé ◽

F. Marceau ◽

B. Thériault ◽

J.-N. Drouin ◽

D. Regoli

Keyword(s):

Direct Action ◽

Smooth Muscles ◽

Hypotensive Effect ◽

Rabbit Heart ◽

Vascular Effects ◽

Large Arteries ◽

Endogenous Prostaglandins ◽

Specific Receptors

The results obtained in this study indicate that the complex cardiovascular effect of BK in vivo may result from direct actions on vascular smooth muscles, presumably mediated by at least two types of receptors, as well as from the release of endogenous prostaglandins. BK and congeners exert a direct action on vascular smooth muscle by stimulating specific receptors both of the B1 type (in the aorta, the large arteries, and the mesenteric vein) and of the B2 type (in the jugular vein); and these vascular tissues provide useful preparations for pharmacological studies of bradykinins. Isolated organs perfused through their main arteries with physiological medium respond to BK by an increase of perfusion pressure (vasoconstriction in isolated ears and kidneys) or by a decrease (vasodilation in the rabbit heart). The vascular effects of BK in the heart and the kidney depend in part on the release of endogenous prostaglandins and on the activation of receptors that appear to be of the B2 type.Like other endogenous hypotensive agents, BK appears to reduce the tonus of the peripheral vessels, while contracting large arteries and veins. The results obtained in vitro are discussed with respect to the hypotensive effect in vivo and to the role of kinins in inflammation and oedema.

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Mechanisms responsible for the in vitro relaxation of a novel dibenzothiepine derivative (NSU-242) on tracheal and vascular smooth muscles

European Journal of Pharmacology ◽

10.1016/j.ejphar.2004.02.017 ◽

2004 ◽

Vol 488 (1-3) ◽

pp. 191-199 ◽

Cited By ~ 5

Author(s):

Hiroshi Ozaki ◽

Masatoshi Hori ◽

Jiro Takeo ◽

Jun-ichiro Hata ◽

Shuji Jinno ◽

...

Keyword(s):

Smooth Muscles ◽

Vascular Smooth Muscles

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Development of RA101348, a Potent Cyclic Peptide Inhibitor of C5 for Complement-Mediated Diseases

Blood ◽

10.1182/blood.v124.21.2936.2936 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2936-2936 ◽

Cited By ~ 7

Author(s):

Alonso Ricardo ◽

Michelle Arata ◽

Steven J DeMarco ◽

Ketki Dhamnaskar ◽

Robert Hammer ◽

...

Keyword(s):

Protein Interactions ◽

Cyclic Peptide ◽

Atypical Hemolytic Uremic Syndrome ◽

Protein Protein Interactions ◽

Self Administration ◽

Complement Activity ◽

Life Threatening ◽

Activation Pathways

Abstract The involvement of complement in the pathogenesis of serious, life-threatening diseases underscores the importance in developing therapeutics to intervene in this cascade. Specifically, dysregulation of the complement system is central in multiple hematologic conditions. Antibody blockade of the C5 component of complement has been approved as a treatment for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome (aHUS), validating C5 as a therapeutic target for these conditions. Ra Pharma has developed a macrocyclic peptide platform capable of yielding potent inhibitors of protein-protein interactions. Recently, we developed a short cyclic peptide that binds complement C5 with high affinity (KD = 2.6 nM) and inhibits its cleavage into C5a and C5b by a novel mechanism. At low nanomolar concentrations, RA101348 blocks the formation of the terminal complement complex (TCC) in vitro, as demonstrated in RBC lysis assays for both alternative and classical complement activation pathways. Inhibition of in vivo complement activity was evaluated in cynomolgus monkeys following single- and multi-dose subcutaneous (SC) administration. RA101348 exhibited high SC bioavailability, and low, single doses inhibited complement activity by 90-100%. The effect is durable owing to an in vivo terminal half-life of approximately 72 hrs. Repeat dosing for 7 days was well tolerated in monkeys at up to 100-fold above projected human therapeutic doses and resulted in sustained inhibition of complement activity. RA101348 is a novel therapeutic approach for inhibiting the activity of C5 for the treatment of disorders caused or associated with complement dysregulation. It offers the potential to enable self-administration of therapeutics for patients with PNH and other complement disorders. Disclosures Ricardo: Ra Pharmaceuticals: Employment. Arata:Ra Pharmaceuticals: Employment. DeMarco:Ra Pharmaceuticals: Employment. Dhamnaskar:Ra Pharmaceuticals: Employment. Hammer:Ra Pharmaceuticals: Employment. Josephson:Ra Pharmaceuticals: Employment. Seyb:Ra Pharmaceuticals: Employment. Tobe:Ra Pharmaceuticals: Employment. Wang:Ra Pharmaceuticals: Employment. Zheng:Ra Pharmaceuticals: Employment. Treco:Ra Pharmaceuticals: Employment.

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Involvement of the vanilloid receptor 1 in the mechanism of analgetic effect of ketorolac

Acta Facultatis Pharmaceuticae Universitatis Comenianae ◽

10.2478/afpuc-2013-0016 ◽

2013 ◽

Vol 60 (2) ◽

pp. 49-55

Author(s):

Yadlovskyi O. ◽

Bukhtiarova T. ◽

Bobkova L. ◽

Tatianchenko I. ◽

Monchak I.

Keyword(s):

Portal Vein ◽

Initial Level ◽

Smooth Muscles ◽

Tail Flick ◽

Active Centre ◽

Vanilloid Receptor 1 ◽

Antinociceptive Effects ◽

Agonist And Antagonist

Abstract The existing ideas about the ketorolac mechanism of analgetic potency are associated with its effect on COX isoenzymes and its significant potency needs to be explained more completely. Its impact on vanilloid receptors 1 (TRPV1) can also be an effective mechanism of analgetic action. We have evaluated the analgetic potential of ketorolac on the basis of its influence on TRPV1. It is known that Tyr511 and Ser512 amino acids are active central components of TRPV1. Thereby the synthesis of the dipeptide Tyr-Ser has been conducted as a model of the TRPV1 active centre. In our model test, using spectrofluorometry, the formation of intermolecular complexes was shown: “ketorolac - Tyr-Ser” and “capsaicin - Tyr-Ser” at stability constants Kr=0.53 • 10-4 L/mol; Kkor= 0.998 and Kr =0.3 • 10-4 L/mol; Kkor=0.998, respectively. Verification of the data was proved in experiments in vitro (the preparation of portal vein) and in vivo (Tail-flick model), with the TRPV1 agonist and antagonist. On portal vein, it was established (from initial level) by Krebs solution that the range of relaxation of smooth muscles of the portal vein (SM) was as follows at the concentration of capsaicin 0.1 μmol/L +30.3%±5.3%, capsazepine 0.5 μmol/L -3.2±2.7% and ketorolac 1.0 μmol/L -60.0±7.0%. In the experiment, at the above mentioned doses of ketorolac on the background of the action of the capsaicin (capsazepine), the range of relaxation of smooth muscles (SM) of the portal vein was -59.3±5.0% and -63.0±6.0% respectively testifying to the fact that the effect of capsaicinoids was neutralised by ketorolac. On the Tail-flick model, an atypical potentiation of the ketorolac antinociception at simultaneous use with the capsaicin was shown. The obtained data allow suggesting that TRPV1 can activate the ketorolac antinociceptive effects.

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