Antiserotonergic properties of terguride in blood vessels, platelets, and valvular interstitial cells.

Serotonin (5-hydroxytryptamine; 5-HT) is involved in heart valve tissue fibrosis, pulmonary arterial fibrosis, and pulmonary hypertension. We aimed at characterizing the antiserotonergic properties of the ergot alkaloid derivative terguride [1,1-diethyl-3-(6-methyl-8α-ergolinyl)urea] by using functional receptor assays and valvular interstitial cell culture. Terguride showed no vasoconstrictor effect in porcine coronary arteries (5-HT(2A) receptor bioassay) and no relaxant effect in porcine pulmonary arteries (5-HT(2B) receptor bioassay). Terguride behaved as a potent antagonist at 5-HT(2A) receptors (noncompetitive antagonist parameter pD'2 9.43) and 5-HT(2B) receptors (apparent pA2 8.87). Metabolites of terguride (N″-monodeethylterguride and 6-norterguride) lacked agonism at both sites. N″-monodeethylterguride and 6-norterguride were surmountable antagonists at 5-HT(2A) receptors (pA2 7.82 and 7.85, respectively) and 5-HT(2B) receptors (pA2 7.30 and 7.11, respectively). Kinetic studies on the effects of terguride in pulmonary arteries showed that the rate to reach drug-receptor equilibrium for terguride was fast. Washout experiments showed that terguride easily disappeared from the receptor biophase. Pretreatment with terguride inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC50 16 nM). In porcine valvular interstitial cells, 5-HT-induced activation of extracellular signal-regulated kinase (ERK) 1/2, an initiator of cellular proliferation and activity, was blocked by terguride as shown by Western blotting. In these cells, the stimulatory effect of 5-HT on [³H]proline incorporation (index of extracellular matrix collagen) was blocked by terguride. Because of the inhibition of both 5-HT(2A) and 5-HT(2B) receptors, platelet aggregation, and cellular proliferation and activity (ERK1/2 phosphorylation and collagen production) terguride may have therapeutic potential in the treatment of fibrotic disorders.

A novel class of nitrovasodilators: potency and in vitro tolerance of organic aminoalkylnitrates.

Experimental studies on organic alkylnitrates have shown that both vasodilator potency and development of in vitro tolerance (tachyphylaxis) correlate with the number of nitrate groups in the molecule. However, introduction of an amino group to ethylnitrate yielded 2-nitrooxyethylammoniumnitrate (1), which is highly active but without inducing in vitro tolerance. Therefore, we prepared a series of aminoalkylnitrates and investigated vasorelaxation and tachyphylaxis on isolated prostaglandin F2alpha-precontracted porcine pulmonary arteries with intact endothelium. Tachyphylaxis was studied by incubating the arteries with EC100 of the respective aminoalkylnitrate and, after a 45-minute washout phase, measuring vasorelaxation again. All of the aminoalkylnitrates caused vasodilation, but the effect did not correlate with the number of nitrate moieties in the molecule. None of the substances was able to outperform compound 1, not even oligonitrates. Generally, structure­activity relationships found for alkylnitrates are obviously not valid for aminoalkylnitrates. Whereas the most potent aminomononitrate 1 evokes no in vitro tolerance, others exhibit tachyphylaxis independently of their vasodilator potency. We have shown that vasorelaxant activity and induction of tachyphylaxis are modulated significantly by introduction of an amino group to the alkylnitrate scaffold. Our results indicate that aminoalkylnitrates have to be considered as an individual class of nitrovasodilators with different structure­activity relationships and vasodilator properties.

Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins.

BACKGROUND: We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca(2+) [Ca(2+)](i). We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca(2+)](i). METHODS: HSV were investigated in vitro either unfrozen within 12h after removal or after storage at -196 degrees C in a cryomedium containing 1.8M dimethyl sulfoxide and 0.1M sucrose as cryoprotectant additives. RESULTS: Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration-response curves for CaCl(2) on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (-)-(R) enantiomer of SDZ 202-791 whereas the Ca(2+) channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl(2) when tested after cryostorage. These functional effects were reflected by changes in [Ca(2+)](i) as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 microM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage. CONCLUSIONS: Data suggested that cryopreservation modified [Ca(2+)](i) of venous smooth muscle cells (1) through depolarization-induced changes in Ca(2+) influx and (2) through activation of Rho kinase signaling pathways.

Characterisation of the relaxant response to raloxifene in porcine coronary arteries.

The present study characterises the vasorelaxant response to raloxifene in isolated rings of porcine coronary artery. Tissues precontracted either with KCl (30 mM) or prostaglandin F(2alpha) (PGF(2alpha); 3 microM) were concentration-dependently relaxed by raloxifene (0.1-10 microM). Relaxation was not inhibited by the estrogen receptor antagonist 7alpha-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17beta-diol (ICI 182,780; 1 microM). Preincubation with raloxifene (1-3 microM) caused an inhibition of the KCl or PGF(2alpha)-induced contraction. The effects of raloxifene were independent of the endothelium. The relaxant response to raloxifene was slow in the onset and could not be reversed after repeated washings. Raloxifene did not affect Ca(2+) release from intracellular stores since it failed to inhibit a transient contraction induced by caffeine (10 mM). Raloxifene-induced relaxation was not influenced by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM; 10-20 microM). Calcium-induced contractions in Ca(2+)-free high K(+) (60 mM) depolarising medium were concentration-dependently inhibited by raloxifene (0.3-3 microM). If arterial rings were incubated with the L-type Ca(2+) channel activator (S)-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester ((S)-(-)-Bay K 8644; 0.1 microM), cumulative concentration-response curves to Ca(2+) were shifted to the left. Raloxifene (0.3-3 microM) inhibited the effect of (S)-(-)-Bay K 8644 in a concentration-dependent manner. 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580; 10 microM), an inhibitor of p38 mitogen-activated protein kinase (MAPK), diminished raloxifene-induced relaxation in endothelium-denuded arterial rings. Western blot analysis demonstrated that raloxifene stimulated p38 MAPK. It is concluded that raloxifene has an inhibitory effect on voltage-gated and receptor-operated L-type Ca(2+) channels in porcine coronary arteries, thus inducing vascular relaxation independent of the endothelium. p38 MAPK is, at least in part, involved in the relaxant response to raloxifene.

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies.

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.

NO-donors, part 9 : diazeniumdiolates inhibit human platelet aggregation and induce a transient vasodilatation of porcine pulmonary arteries in accordance with the NO-releasing rates.

Diazeniumdiolates (NONOates), among them a ciprofloxacin-diazeniumdiolate hybrid compound, were synthesized and the pH-, temperature- and structure-dependent liberation of nitric oxide (NO) was monitored by laser magnetic resonance spectroscopy (LMRS). The compounds induced a transient and reversible relaxation (EC(50) 8.3-150 nM) of pulmonary arteries independently from intact endothelium by stimulation of guanylyl cyclase (sGC). Increase in vascular cGMP was observed and blocking sGC with ODQ, an inhibitor of the NO-sensitive guanylyl cyclase, induced a rightward shift of the concentration-response curves. Repeated exposure did not show homologous desensitization. ADP-induced platelet aggregation (IC(50) = 0.15-3 microM, IC(50) for SNP: 2 microM) and collagen-induced aggregation were potently inhibited. Preincubation with ODQ also diminished these inhibitory effects.

Activation of mast cells induced by agonists of proteinase-activated receptors under normal conditions and during acute inflammation in rats.

Functions of thrombin as a modulator of inflammation and tissue repair are mediated by the proteinase-activated receptor (PAR) family. Some of these effects may be induced by activation of mast cells. To characterize the degranulation of rat peritoneal mast cells in response to PAR agonists, the effects of thrombin, trypsin and peptide agonists of PARs (PAR-AP, proteinase-activated receptor-activating peptides) on secretion were investigated. The release of beta-hexosaminidase by thrombin (0.01-1 microM) was concentration-dependent and mediated via PAR(1), as evidenced by cathepsin G (100 microM)-induced inactivation of PAR(1) and thrombin-stimulated PAR(1) desensitization. Trypsin (1 microM) accelerated histamine secretion. The PAR(1)-AP, TRAP (SFFLRN, 1-100 microM) and the PAR(2)-AP SLIGRL (5-100 microM) caused the release of histamine, and beta-hexosaminidase from inflammatory mast cells were obtained from a model of acute peritonitis in rats. Relative to the response to compound 48/80, the thrombin- and TRAP-induced release of beta-hexosaminidase was higher in inflammatory mast cells than in the control. This suggests that additional exposure of PAR(1) on mast cells to PAR agonists or an increase in PARs sensitivity to PAR agonists probably occurred during acute inflammation.

5-HT7, but not 5-HT2B, receptors mediate hypotension in vagosympathectomized rats.

This study evaluated the possible involvement of 5-HT(2B) receptors in long-lasting hypotension to 5-hydroxytryptamine (5-HT), which is predominantly mediated by 5-HT7 receptors, in anaesthetised vagosympathectomized rats. Intravenous injections of 5-HT and 5-carboxamidotryptamine (5-CT) elicited a dose-dependent hypotension that was dose-dependently antagonised by (R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl) ethyl] pyrrolidine (SB-269970; a selective 5-HT7 receptor antagonist), but not by saline. Interestingly, alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine (BW723C86; a 5-HT(2B) receptor agonist) produced vasopressor responses without affecting hypotension to 5-HT. These results suggest that hypotension to 5-HT and 5-CT is mainly mediated by 5-HT7 receptors, whilst the role of 5-HT(2B) receptors seems unlikely.

The thrombin inhibitor argatroban does not influence the endothelium-dependent relaxant and contractile responses of isolated rabbit carotid arteries.

Atherosclerotic endothelial dysfunctions are associated with a reduced NO production, which is probably due to impaired NO synthase (eNOS) activity or a deficiency of the substrate L-arginine. In the present studies, the influence of argatroban on isolated rabbit carotid arteries was investigated to determine whether the arginine derivative argatroban can improve the endothelium-dependent relaxation. Rings from rabbit carotid arteries were placed in 10 ml organ baths for isometric tension recording. Endothelial integrity was assessed by the acetylcholine-induced relaxation of PGF2alpha-precontracted rings; after mechanical removal of the endothelium the relaxation was abolished. Preincubation of the vessels in vitro with L-NAME, an inhibitor of the eNOS, diminished significantly the acetylcholine-induced relaxation by more than 50%. After i.v. application of L-NAME (100 mg/kg) in rabbits, relaxation in response to acetylcholine was significantly reduced compared to the control when the vessels were studied ex vivo in an organ bath. The contractile effects of phenylephrine and 5-HT were slightly enhanced. Argatroban is a selective, potent, synthetic thrombin inhibitor; after i.v. application at doses of 0.5 and 1.0 mg/kg, a significant prolongation of the plasma coagulation time (measured as thrombin time and a PTT) of up to 60 min was found in rabbits. In vitro argatroban did not affect the acetylcholine-induced relaxation or the contractile response to phenylephrine and 5-HT. After i.v. application, the ex vivo experiments in the organ bath showed that after 30 min the relaxant responses of the carotid arteries to acetylcholine and the contractile effects of phenylephrine and 5-HT were not influenced by pretreatment with argatroban. The present studies suggest that argatroban has no vascular effects in vitro and ex vivo in normal rabbits.

NO donors. Part 16: investigations on structure-activity relationships of organic mononitrates reveal 2-nitrooxyethylammoniumnitrate as a high potent vasodilator.

The vasoactive properties of 14 organic mononitrates were investigated in vitro using PGF(2alpha)-precontracted porcine pulmonary arteries. A surprisingly wide range of vasorelaxant potencies was observed (pD(2): 3.36-7.50). Activities showed to be highly sensitive to the molecular structure and the substituents at the molecular carrier of the nitrate group. A correlation between lipophilicity and vasorelaxant potency could not be recognized. 2-Nitrooxyethylammoniumnitrate (1) was found to be slightly superior to the high potency trinitrate GTN.

Potency and in vitro tolerance of organic nitrates: partially denitrated metabolites contribute to the tolerance-devoid activity of pentaerythrityl tetranitrate.

Neither therapeutic dosage of nitrovasodilators nor the development of tolerance correlates with nitrate groups in these molecules. Clinically, low dosages of glyceryl trinitrate (GTN) develop tolerance, but 100-fold higher dosages of pentaerythrityl tetranitrate (PETN) do not. Vasorelaxation was studied on prostaglandian F2alpha (PGF2alpha)-precontracted porcine pulmonary arteries in organ bath procedure. In vitro tolerance was induced by incubating the arteries with different nitrate concentrations and thereafter concentration-response curves were repeated. Furthermore, 14 mg/kg PETN were daily administered to rats by gavage; PETN and metabolites were measured in feces and blood. In vitro, the vasodilator potencies increased from mononitrates to tetranitrates (pD2: 4.14 to 8.18); PETN was the most potent vasodilator. In vitro tolerance was found with PETN and trinitrates but not with dinitrates and mononitrates. Thus, in vitro tolerance correlated with the in vitro potency of nitrates but not with the vasodilator potency of NO donors in general, because S-nitroso-N-aectyl-D-penicillamine and N-phenylpiperazin-NONOate were more potent than GTN but did not induce tolerance. After feeding of rats with PETN, pentaerythrityl dinitrate (PEdiN) and mononitrate (PEmonoN) but neither PETN nor PEtriN (both detected in feces) were found in the blood. The missing systemic bioavailability of PETN and PEtriN may explain the discrepancy between in vitro and in vivo findings. We conclude that the partially denitrated metabolites PEdiN and PEmonoN contribute to the moderate and tolerance-devoid clinical activity of PETN.

Impact of freezing/thawing procedures on the post-thaw viability of cryopreserved human saphenous vein conduits.

BACKGROUND: Cryopreserved human blood vessels are important tools in reconstructive surgery. However, patency of frozen/thawed conduits depends largely on the freezing/thawing procedures employed. METHODS: Changes in tone were recorded on rings from human saphenous vein (SV) and used to quantify the degree of cryoinjury after different periods of exposure at room temperature to the cryomedium (Krebs-Henseleit solution containing 1.8M dimethyl sulfoxide and 0.1M sucrose) and after different cooling speeds and thawing rates following storage at -196 degrees C. RESULTS: Without freezing, exposure of SV to the cryomedium for up to 240 min did not modify contractile responses to noradrenaline (NA). Pre-freezing exposure to the cryomedium for 10-120 min attenuated significantly post-thaw maximal contractile responses to NA, endothelin-1 (ET-1) and potassium chloride (KCl) by 30-44%. Exposure for 240 min attenuated post-thaw contractile responses to all tested agents markedly by 62-67%. Optimal post-thaw contractile activity was obtained with SV frozen at about -1.2 degrees C/min and thawed slowly at about 15 degrees C/min. In these SV maximal contractile responses to NA, ET-1 and KCl amounted to 66%, 70% and 60% of that produced by unfrozen controls. Following cryostorage of veins for up to 10 years the responsiveness of vascular smooth muscle to NA was well maintained. CONCLUSION: Cryopreservation allows long-term banking of viable human SV with only minor loss in contractility.

Synthesis and vasorelaxant properties of hybrid molecules out of NO-donors and the beta-receptor blocking drug propranolol.

S-Nitroso-N-acetylpenicillamine (SNAP) and 3-nitrooxypivaloyl acid were combined in the form of the respective amides with propranolol, in order to obtain prodrugs (NO-propranololes) with beta-receptor blocking properties of the latter compound with nitric oxide releasing properties of the former compounds. A respective nitratoester could not be synthesized, because it immediately rearranges to the amide after deprotection of the amino group. In vitro tests on porcine pulmonary arteries showed that both types of hybrid molecules (6, 12) elicited vasorelaxation, but the nitratoamide was less potent by more than one order of magnitude. The vasorelaxant effect of SNAP was more pronounced than that of the SNAP-hybrid (12), on the other hand the nitratoamide 6 was more potent than 3-nitrooxypivaloyl acid.

Fusion proteins with anticoagulant and fibrinolytic properties: functional studies and structural considerations.

In an effort to combine the benefits of fibrinolytics, such as staphylokinase, with those of thrombin inhibitors for the prevention of vessel reocclusion after vascular injury, we have produced several chimeric proteins with plasminogen-activating and thrombin-inhibiting properties. Fusion proteins were constructed consisting of the modules staphylokinase (Sak), the factor Xa cleavage site, and various dipetalin (Dip) domains (H(6)-Sak-Dip-I+II, H(6)-Sak-Dip-I, and H(6)-Sak-Dip-II). Sak stimulates fibrinolysis via activation of plasminogen, whereas dipetalin is a two-domain, Kazal-type inhibitor of thrombin. NMR spectroscopy of the fusion proteins revealed that the molecular structures of the modules are retained in the fusion protein and that no significant interactions occur between the modules in terms of their functionally relevant regions. In enzymatic thrombin inhibition tests and blood coagulation assays (thrombin, prothrombin, and activated partial thromboplastin times), no significant differences in anticoagulant capacity were observed between the fusion protein H(6)-Sak-Dip-I+II and isolated Dip-I+II, even at nanomolar concentrations. Similar results (i.e., the inhibition of thrombin-induced platelet aggregation and the inhibition of thrombin-induced vascular relaxation) were obtained when the cellular thrombin effects were studied. The fusion protein containing Dip-I has less but still significant thrombin inhibitory effects compared with those of H(6)-Sak-Dip-I+II. In contrast, the H(6)-Sak-Dip-II protein failed to inhibit thrombin in each of the assays used. The plasminogen-activating and fibrinolytic activities of the fusion proteins are similar to those of wild-type Sak. The individual dipetalin domains do not activate plasminogen. In conclusion, the fusion protein H(6)-Sak-Dip-I+II is a bifunctional molecule able to activate fibrinolysis via plasminogen activation and inhibit blood coagulation via direct inhibition of thrombin.