Rodolfo Pedro Rothlin

Bradykinin B1 receptors in human umbilical vein.

The present study was undertaken to demonstrate the presence of bradykinin B1 receptors mediating contraction of human umbilical vein. The bradykinin B1 receptor selective agonist, des-Arg9-bradykinin, produced a dose-dependent contractile response of human umbilical vein rings. Furthermore, des-Arga-bradykinin-mediated response increased in a time-dependent manner in vitro. The maximal response to des-Arg9-bradykinin, expressed as percentage of the maximum elicited by serotonin, was: 10 +/- 2 at 15 min, 55 +/- 5 at 120 min and 80 +/- 3 at 300 min. Des-Arg9-bradykinin-mediated contractions were inhibited by the specific bradykinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin which produced parallel shifts in the dose-response curve to the selective bradykinin B1 receptor agonist. Schild regression analysis of data established a pA2 value of 6.16 +/- 0.06. Kinin-induced contraction was not modified by pre-treatment with indomethacin (10 microM), a cyclo-oxygenase inhibitor. On the other hand, continuous exposure to the anti-inflammatory steroid dexamethasone (100 microM) or to the protein synthesis inhibitor cycloheximide (70 microM) largely prevented the sensitization to des-Arg9-bradykinin in incubated human umbilical vein rings. These results confirm the presence of bradykinin B1 receptors which mediate contraction in isolated human umbilical vein. These responses are up-regulated in a time- and protein synthesis-dependent process.

Bradykinin B1 receptors in human umbilical vein: pharmacological evidence of up-regulation, and induction by interleukin-1β

Bradykinin B1 receptor-mediated responses increase as a function of in vitro incubation in the human umbilical vein. When tissues were continuously treated with the protein synthesis inhibitor, cycloheximide, or with the protein trafficking inhibitor, brefeldin A, pEC50 and maximal response to the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin, were significantly diminished. The anti-inflammatory steroid, dexamethasone, produced a rightward shift of the concentration-response curve to des-Arg9-bradykinin, without affecting the maximal response. Furthermore, lipopolysaccharide or recombinant human interleukin-1 beta potentiate the bradykinin B1-sensitized responses, showing a leftward shift of the concentration-response curve to des-Arg9-bradykinin, without modifying the maximal response. On the other hand, bradykinin B2 receptor-mediated responses were unaffected by continuous exposure to cycloheximide, dexamethasone or lipopolysaccharide. These results provide pharmacological evidence to support the view that the de novo synthesis of bradykinin B1 receptors is involved in the induction of vascular responses in the human umbilical vein. This up-regulation process seems to be selective for bradykinin B1 receptors. The inhibitory effect of dexamethasone and the potentiating actions of lipopolysaccharide and exogenous human recombinant interleukin-1 beta on des-Arg9-bradykinin-mediated responses, suggest the possible role of interleukin-1 beta in the bradykinin B1 receptor up-regulation phenomenon in human umbilical vein.

Characterization of α1‐adrenoceptor subtypes mediating vasoconstriction in human umbilical vein

The present study attempted to characterize pharmacologically the subtypes of α‐adrenoceptors mediating contractions in human umbilical vein (HUV). HUV rings were mounted in isolated organ baths and cumulative concentration‐response curves were constructed for the α‐adrenoceptor agonists phenylephrine and adrenaline. Adrenaline was more potent than phenylephrine (pD2=7.29 and 6.04 respectively). Isoproterenol exhibited no agonism on KCl pre‐contracted HUV rings. Propranolol (1 μM) and rauwolscine (0.1 μM) did not affect the concentration‐response curves to adrenaline. These results demonstrate the lack of involvement of functional β‐ or α2‐adrenoceptors in adrenaline‐induced vasoconstriction. The non subtype selective α1‐adrenoceptor antagonist prazosin was evaluated on phenylephrine and adrenaline concentration‐response curves. The effects of the competitive α1A and α1D‐adrenoceptor antagonists, 5‐methyl urapidil and BMY 7378 and the irreversible α1B selective compound chloroethylclonidine (CEC) were also evaluated on adrenaline concentration‐response curves. The potencies of prazosin against responses mediated by adrenaline (pA2=10.87) and phenylephrine (pA2=10.70) indicate the involvement of prazosin‐sensitive functional α1‐adrenoceptor subtype in vasoconstriction of the HUV. The potencies of 5‐methyl urapidil (pA2=6.70) and BMY 7378 (pA2=7.34) were not consistent with the activation of an α1A‐ or α1D‐adrenoceptor population. Exposure to a relatively low CEC concentration (3 μM) abolished the maximum response to adrenaline suggesting that this response was mediated by an α1B‐adrenoceptor subtype. We conclude that HUV express a prazosin‐sensitive functional α1‐adrenoceptor resembling the α1B‐subtype according with the low pA2 values for both 5‐methyl urapidil and BMY 7378 and the high sensitivity to CEC.

Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein

This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating contraction in human umbilical vein (HUV). HUV rings were mounted in organ baths and concentration–response curves to U‐46619 (TXA2 mimetic) were constructed in the absence or presence of SQ‐29548 or ICI‐192,605 (TP receptor antagonists). U‐46619 was a potent constrictor (pEC50: 8.03). SQ‐29548 and ICI‐192,605 competitively antagonized responses to U‐46619 with pKB values of 7.96 and 9.07, respectively. Concentration–response curves to EP receptor agonists: PGE2, misoprostol and 17‐phenyl‐trinor‐PGE2 gave pEC50 values of 5.06, 5.25 and 5.32, respectively. Neither pEC50 nor maximum of PGE2 and 17‐phenyl‐trinor‐PGE2 concentration–response curves were modified by the DP/EP1/EP2 receptor antagonist AH 6809 (1 μM). However, ICI‐192,605 produced a concentration‐dependent antagonism of the responses to all the EP receptor agonists. The pA2 estimated for ICI‐192,605 against PGE2 or misoprostol were 8.91 and 9.22, respectively. Concentration–response curves to FP receptor agonists: PGF2α and fluprostenol gave pEC50 values of 6.20 and 5.82, respectively. ICI‐192,605 (100 nM) was completely ineffective against PGF2α or fluprostenol. In addition, lack of antagonistic effect of AH 6809 (1 μM) against PGF2α was observed. In conclusion, the findings obtained with TP‐selective agonist and antagonists provide strong evidence of the involvement of TP receptors promoting vasoconstriction in HUV. Furthermore, the action of the natural and synthetic EP receptor agonists appears to be mediated via TP receptors. On the other hand, the results employing FP receptor agonists and antagonists of different prostanoid receptors suggest the presence of FP receptors mediating vasoconstriction in this vessel.

Pharmacological characterization of muscarinic receptor subtypes mediating vasoconstriction of human umbilical vein

The present study attempted to pharmacologically characterize the muscarinic receptor subtypes mediating contraction of human umbilical vein (HUV). HUV rings were mounted in organ baths and concentration–response curves were constructed for acetylcholine (ACh) (pEC50: 6.16±0.04; maximum response 80.00±1.98% of the responses induced by serotonin 10 μM). The absence of endothelium did not modify the contractile responses of ACh in this tissue. The role of cholinesterases was evaluated: neither neostigmine (acetylcholinesterase inhibitor) nor iso‐OMPA (butyrylcholinesterase inhibitor) modified ACh responses. When both enzymes were simultaneously inhibited, a significantly but little potentiation was observed (control: pEC50 6.33±0.03; double inhibition: pEC50 6.57±0.05). Atropine, nonselective muscarinic receptors antagonist, inhibited ACh‐induced contraction (pKB 9.67). The muscarinic receptors antagonists pirenzepine (M1), methoctramine (M2) and pFHHSiD (M3) also antagonized responses to ACh. The affinity values estimated for these antagonists against responses evoked by ACh were 7.58, 6.78 and 7.94, respectively. On the other hand, PD 102807 (M4 selective muscarinic receptors antagonist) was ineffective against ACh‐induced contraction. In presence of a blocking concentration of pirenzepine, pFHHSiFD produced an additional antagonism activity on ACh‐induced responses. The M1 muscarinic receptors agonist McN‐A‐343 produced similar maximum but less potent responses than ACh in HUV. The calculated pA2 for pirenzepine against McN‐A‐343 induced responses was 8.54. In conclusion, the data obtained in this study demonstrate the role of M1 muscarinic receptor subtypes and suggest the involvement of M3 muscarinic receptor subtypes in ACh‐induced vasoconstriction in HUV rings. In addition, the vasomotor activity evoked by ACh does not seem to be modulated by endothelial factors, and their enzymatic degradation appears to have little functional relevance in this tissue.

EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin

INTRODUCTION This study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE(2)-mediated TP receptor contraction of human arteries was also investigated. MATERIALS AND METHODS Fifty-two type-2 diabetics with CAD on chronic aspirin (100 mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100 nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE(2). RESULTS Arachidonic acid (1 mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63 ± 4 AU), which was significantly reduced by in vitro exposure to EV-077 (38 ± 3 AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE(2) (P<0.01). CONCLUSIONS Aspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE(2) was effectively inhibited by EV-077.

Characterization of 5-HT receptor subtypes mediating contraction in human umbilical vein. 2. Evidence of involvement of 5-HT1B receptors using functional studies

Abstract. Previous studies have shown that a heterogeneous 5-HT receptor population may be involved in vasoconstrictor actions of 5-HT in human umbilical vein (HUV). The aim of the present study was to evaluate whether the 5-HT1B/1D receptor subtype mediates contraction in this tissue.5-HT1B/1D-mediated responses can be enhanced or unmasked after exposure to threshold or sub-threshold KCl concentrations. In HUV rings, when 5-HT, α-Me-5HT or bradykinin concentration-response curves (CRC) were generated in the presence or absence of sub-threshold concentrations of KCl, there were not significant differences between the control and the treated rings. On the other hand, sumatriptan, the classic selective 5-HT1B/1D receptor agonist, produced a concentration-related contraction that was potentiated in the presence of sub-threshold KCl concentration. In addition, L-694,247, the novel selective 5-HT1B/1D receptor agonist, displayed a concentration-dependent contraction with high potency in HUV. The presence of sub-threshold concentrations of KCl produced a marked leftward shift of its CRCs.GR-55562, a 5-HT1B/1D-selective antagonist, competitively blocked sumatriptan CRCs with an estimated pA2 of 8.00 and a slope not different from unity. Likewise, SB-216641, a selective 5-HT1B antagonist, produced a parallel rightward shift of sumatriptan CRCs in HUV. The Schild analysis yielded a pA2 of 9.29, with a slope not different from unity. In addition, L-694,247 contractile responses were competitively blocked by SB-216641 with an estimated pA2 value of 9.12 and a Schild slope not different from unity. On the other hand, ketanserin behaved as a weak antagonist of L-694,247-induced responses, yielding a calculated pA2 value of 6.40.In summary, the results obtained in this study support that the 5-HT1B receptor subtype is involved in vasoconstrictor responses in HUV.

Expression and functional evidence of the prostaglandin F2α receptor mediating contraction in human umbilical vein

Our purposes were to perform the pharmacological characterization of PGF(2alpha) receptor (prostanoid FP-receptor) involved in human umbilical vein contraction and confirm its expression in this tissue. Umbilical cords from healthy patients after full-term deliveries were employed. The vein was dissected out of cords and used for either isolated organ bath or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. The natural prostanoid FP-receptor agonist, PGF(2alpha), and its selective analogues, latanoprost and bimatoprost free acids are full agonists (produce more than 80% of the maximal contractile response to 5-HT) in human umbilical vein. The agonist potency (pEC(50)) order was PGF(2alpha) (6.01+/-0.05)>latanoprost free acid (5.65+/-0.07)=bimatoprost free acid (5.59+/-0.08). The contractile effects of PGF(2alpha) and latanoprost free acid were blocked competitively by the prostanoid FP-receptor antagonist, AL-8810. The antagonist potencies (pK(B)) of AL-8810 vs. PGF(2alpha) (5.93+/-0.05) and vs. latanoprost free acid (6.40+/-0.08) in human umbilical vein are in good agreement with its ability to antagonize prostanoid FP receptors of rat, mouse and human cells. In all samples, clear signal was detected for cDNA amplification of prostanoid FP receptor and the specific prostanoid FP-receptor antibody recognized a protein of approximately 64 kDa. In conclusion, taking into account the obtained functional and biochemical data, we propose for the first time that human umbilical vein express prostanoid FP-receptors and these receptors could be involved in the vasoconstriction action of PGF(2alpha) in this tissue.

Endothelial angiotensin-converting enzyme and neutral endopeptidase in isolated human umbilical vein: an effective bradykinin inactivation pathway.

Kinins are metabolized by metallopeptidases present in different tissues. The aim of this study was to evaluate, employing functional studies in isolated human umbilical vein, the possible participation of angiotensin-converting enzyme, neutral endopeptidase and aminopeptidase P as an inactivation pathway of bradykinin, as well as assess if the endothelial layer is involved in this process. Concentration-response curves to bradykinin were constructed after 120 min incubation period on human umbilical vein rings with and without endothelium and enzymatic inhibitors were applied 30 min before construction of concentration-response curves. The presence of endothelium was confirmed by histological studies. Bradykinin-induced contractile responses were potentiated in human umbilical vein without endothelium when compared to intact tissues. Application of captopril 1 μM (angiotensin-converting enzyme inhibitor) or phosphoramidon 10 μM (neutral endopeptidase inhibitor) induced a leftward shift of bradykinin-elicited responses in human umbilical vein with endothelium while no effect was observed in tissues denuded of endothelium under the same treatment. Exposure to apstatin 10 μM (aminopeptidase P inhibitor) did not potentiate bradykinin-induced effects in intact human umbilical vein. When angiotensin-converting enzyme and neutral endopeptidase were concomitantly inhibited, there was a higher potentiation of bradykinin-elicited responses compared to the effects observed under individual inhibition of either enzyme. Moreover, concentration-response curves to FR190997, a non-peptidic bradykinin B(2) receptor agonist, were not modified under dual enzymatic inhibition. In conclusion, our results demonstrate for the first time the functional relevance of angiotensin-converting enzyme and neutral endopeptidase, localized on the endothelial layer, acting concurrently as a bradykinin inactivating pathway in isolated human umbilical vein.

Neutral Endopeptidase Up-Regulation in Isolated Human Umbilical Artery: Involvement in Desensitization of Bradykinin-Induced Vasoconstrictor Effects

Previous reports show that bradykinin B2 receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.