Effect of warming and flow rate conditions of blood warmers on red blood cell integrity.

BACKGROUND AND OBJECTIVES: Fluid warmers are routinely used to reduce the risk of hypothermia and cardiac complications associated with the infusion of cold blood products. However, warming blood products could generate haemolysis. This study was undertaken to compare the impact of temperature of blood warmers on the per cent haemolysis of packed red blood cells (RBCs) heated at different flow rates as well as non-flow conditions. MATERIALS AND METHODS: Infusion warmers used were calibrated at 41·5°C ± 0·5°C and 37·5°C ± 0·5°C. Cold RBC units stored at 4°C in AS-3 (n = 30), aged 30-39 days old, were divided into half units before being allocated under two different scenarios (i.e. infusion pump or syringe). RESULTS: Blood warmers were effective to warm cold RBCs to 37·5°C or 41·5°C when used in conjunction with an infusion pump at flow rate up to 600 ml/h. However, when the warmed blood was held in a syringe for various periods of time, such as may occur in neonatal transfusions, the final temperature was below the expected requirements with measurement as low as 33·1°C. Increasing the flow with an infusion pump increased haemolysis in RBCs from 0·2% to up to 2·1% at a flow rate of 600 ml/h regardless of the warming device used (P < 0·05). No relevant increase of haemolysis was observed using a syringe. CONCLUSIONS: The use of a blood warmer adjusted to 41·5°C is probably the best choice for reducing the risk of hypothermia for the patient without generating haemolysis. However, we should be cautious with the use of an infusion pump for RBC transfusion, particularly at high flow rates.

Neuroprotective effects of a postischemic treatment with a bradykinin B2 receptor antagonist in a rat model of temporary focal cerebral ischemia.

Bradykinin, an endogenous nonapeptide produced by activation of the kallikrein-kinin system, promotes neuronal tissue damage as well as disturbances in blood-brain barrier function through activation of B2 receptors. In a rat model of focal cerebral ischemia, blockade of B2 receptors before initiation of ischemia with the B2 receptor antagonist, LF 16-0687 Ms, afforded substantial neuroprotection. In order to assess the potential clinical value of this approach, we evaluated the effect of LF 16-0687 Ms given at reperfusion following focal cerebral ischemia on local cerebral blood flow (LCBF), neurological outcome, and infarct size. Sprague-Dawley rats were subjected to MCA occlusion for 90 min by an intraluminal filament. Animals were assigned to one of four treatment arms (n = 7 each): (1) vehicle, (2) LF 16-0687 Ms (1.0 mg/kg/day), (3) LF 16-0687 Ms (3.0 mg/kg/day), or (4) LF 16-0687 Ms (10.0 mg/kg/day) given at reperfusion and repetitively over 2 days. Neurological recovery was examined daily, and infarct volume was assessed histologically on day 7 after ischemia. Physiological parameters and local CBF were not influenced by the treatment. Significant improvement of neurological outcome was observed on postischemic day 3 in animals receiving 1.0 and 3.0 mg/kg/day of LF 16-0687 Ms (P < 0.05). Inhibition of B2 receptors significantly reduced infarct volume in all treated animals predominantly in the cortex. B2 receptor blockade with LF 16-0687 Ms showed neuroprotective effectiveness even when therapy was initiated upon reperfusion, i.e. 90 min after induction of ischemia. Therefore, blockade of B2 receptors seems to be a promising therapeutic approach after focal cerebral ischemia, which deserves further experimental and clinical evaluation.

Effect of a long-term treatment with lovastatin or fenofibrate on hepatic and cardiac ubiquinone levels in cardiomyopathic hamster.

This study was undertaken to determine if long-term oral administration of lovastatin (50 mg/kg per day) or fenofibrate (200 mg/kg per day) was affecting ubiquinone levels in the heart and the liver of cardiomyopathic hamsters. After 23 weeks of treatment, ubiquinone concentrations (CoQ9 + CoQ10) and ubiquinone ratio (CoQ10/CoQ9) were determined in the heart and in the liver. Our results indicate that lovastatin significantly decreased ubiquinone concentrations in the heart (-33%, P < 0.01) but not in the liver (-23%, NS) when compared to controls, whereas fenofibrate did not alter these parameters. Ubiquinone homologues were not equally decreased during lovastatin treatment: the ratio between CoQ10 and CoQ9 was significantly lowered in the heart (-33%, P < 0.001) and in the liver (-75%, P < 0.001) of lovastatin-treated animals. These results suggest that 3-hydroxymethylglutaryl-coenzyme A reductase inhibition (HMG-CoARI) associated with lovastatin treatment in cardiomyopathic hamsters is more marked in the liver than in the heart, while ubiquinone concentrations are more decreased in cardiac than in hepatic tissues. Our data also showed that fenofibrate had no effect on ubiquinone levels.

Synthesis and biological evaluation of bradykinin B(1)/B(2) and selective B(1) receptor antagonists.

We recently described a potent bradykinin B(2) receptor agonist (JMV1116) obtained by replacing the D-Tic-Oic dipeptide moiety of HOE140 by a (3S)-amino-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety. This compound inhibited the specific binding of [(3)H]BK on membranes of CHO cells expressing the human cloned B(2) receptor with nanomolar affinity and contracted both isolated rat uterus and human umbilical vein. These data demonstrated that D-BT could be a good mimic of the Pro-Phe dipeptide. In the present study we characterized B(1) receptor antagonists containing the D-BT moiety. We prepared an analogue of compound JMV1116 deleting the C-terminal arginine residue. The resulting compound (1) had an affinity of 83 nM for the human cloned B(1) receptor. The most remarkable property of 1 is its ability to bind also the B(2) receptor with an affinity of 4.4 nM despite the absence of the C-terminal arginine residue. Modifications at the N-terminal part of 1 associated with the substitution of the thienylalanine residue by alpha-(2-indanyl)glycine resulted in analogues selectively binding to the B(1) receptor with an affinity in the picomolar range.

A rational approach to the design and synthesis of a new bradykinin B(1) receptor antagonist.

We have previously synthesized a potent and selective B(1) bradykinin receptor antagonist, JMV1645 (H-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-BT-OH), containing a dipeptide mimetic ((3S)-amino-5-carbonylmethyl-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety) at the C-terminal. Analogues of this potent B(1) bradykinin receptor antagonist in which the central Pro(2)-Hyp(3)-Gly(4)-Igl(5) tetrapeptide has been replaced by constrained N-1-substituted-1,3,8-triazaspiro¿4. 5decan-4-one ring system were synthesized. Among these analogues, compound JMV1640 (1) was found to have an affinity of 24.10 +/- 9.48 nM for the human cloned B(1) receptor. It antagonized the ¿des-Arg(10)-kallidin-induced contraction of the human umbilical vein (pA(2) = 6.1 +/- 0.1). Compound 1 was devoid of agonist activity at the kinin B(1) receptor. Moreover, it did not bind to the human cloned B(2) receptor. Therefore, JMV1640 constitutes a lead compound for the rational search of nonpeptide B(1) receptor analogues based on the BK sequence.

Design and synthesis of potent bradykinin agonists containing a benzothiazepine moiety.

A bradykinin analogue (H-Arg-Pro-Pro-Gly-Phe-Ser-D-BT-Arg-OH, 3) in which the Pro-Phe dipeptide was replaced by the (3S)[amino]-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety has been synthesized. The same modification was performed on the potent bradykinin B(2) receptor antagonist HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH), in which the -D-Tic-Oic- moiety was replaced by D-BT to yield H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-BT-Arg-OH, 1 (JMV1116). These compounds were examined in vitro for their binding affinity toward bradykinin B(1) and B(2) receptors as well as for their ability to interfere with bradykinin-induced contraction of both human umbilical vein and rat uterus. The two compounds 3 and 1 competed with [(3)H]bradykinin binding to the human cloned B(2) receptor giving K(i) values of 13 +/- 2 and 0.7 +/- 0.1 nM, respectively. Unexpectedly, both compounds were full bradykinin B(2) receptor agonists on the human umbilical vein (pD(2) = 6.60 +/- 0.07 for 3 and 6.80 +/- 0.08 for 1) and rat uterus (pD(2) = 7.20 +/- 0.09 for 3 and 7.50 +/- 0.09 for 1) preparations with the same efficacy as bradykinin. In addition 1 induced a concentration-dependent phosphoinositide production in CHO cells expressing the human cloned B(2) receptor. These data provide evidence for a bioactive conformation of bradykinin constrained at the dipeptide Pro-Phe.

Synthesis and characterization of bradykinin B(2) receptor agonists containing constrained dipeptide mimics.

We have previously shown that substitution of the D-Tic-Oic dipeptide by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety in the bradykinin B(2) receptor antagonist HOE 140 resulted in a full potent and selective bradykinin B(2) receptor agonist (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-D-BT-Arg-OH, JMV1116) exhibiting a high affinity for the human receptor (K(i) 0.7 nM). In the present study, we have investigated the effects of replacement of the D-Tic-Oic moiety by various constrained dipeptide mimetics. The resulting compounds were tested for their binding affinity toward the cloned human B(2) receptor and for their functional interaction with the bradykinin-induced contraction of isolated human umbilical vein. Subsequently, we have designed novel bradykinin B(2) receptor agonists which are likely to be resistant to enzymatic cleavage by endopeptidases and which might represent interesting new pharmacological tools. In an attempt to increase the potency of compound JMV1116, both its N-terminal part and the D-BT moiety were modified. Substitution of the D-arginine residue by a L-lysine residue led to a 10-fold more potent bradykinin B(2) ligand [compound 22 (JMV1465) (K(i) 0.07 nM)], retaining full agonist activity on human umbilical vein. Substitution of the D-BT moiety by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-8-methyl-1, 5-benzothiazepin-4(5H)-one [D-BT(Me)] moiety led to compound 23 (JMV1609) which exhibited a higher agonist activity (pD(2) = 7.4) than JMV1116 (pD(2) = 6.8).

Specific nonpeptide photoprobes as tools for the structural study of the angiotensin II AT(1) receptor.

The aim of this work was to obtain photoactivatable nonpeptide antagonists of the angiotensin II AT(1) receptor. Based on structure-function relationships, two chemical structures as well as appropriate synthetic schemes were chosen as a frame for the design of radiolabeled azido probes. The feasibility of the strategy was first assessed by the synthesis of two tritiated ligands 21 and 22 possessing a high affinity for the AT(1) receptor and a low nonspecific binding to membrane or cell preparations. We then prepared two unlabeled azido derivatives 7 and 14 which retained a fairly high affinity for the AT(1) receptor. The latter compound proved to be suitable for receptor irreversible labeling and was prepared in its tritiated form 28. This tritiated azido nonpeptide probe displayed a K(d) value of 11.8 nM and a low nonspecific binding. It was suitable for specific and efficient covalent labeling of the recombinant AT(1A) receptor stably expressed in CHO cells. The electrophoretic pattern of the specifically labeled entity was strictly identical to that of purified receptor photolabeled with a biotinylated peptidic photoactivatable probe. This new tool should be useful for the mapping of the nonpeptide receptor binding site. These potential applications are discussed in light of the current knowledge of molecular mechanisms of G-protein coupled receptor activation and inactivation.

Omega-conotoxin GVIA is a potent inhibitor of sympathetic neurogenic responses in rat small mesenteric arteries.

1. We have investigated the effects of the N-type calcium channel blocker, omega-conotoxin GVIA, on contractile responses to nerve stimulation, noradrenaline and KCl in rat small mesenteric arteries. In separate experiments, single and summated excitatory junctional potentials (e.j.ps) evoked by nerve stimulation were recorded with an intracellular electrode in the absence and presence of omega-conotoxin. 2. Electrical field stimulation of intramural sympathetic nerves (30 V; 0.25 ms pulse width; 3 s train length; 4-24 Hz) caused frequency-dependent contractions. Cumulative concentration-response curves for the contractions induced by noradrenaline and KCl were constructed in the same preparations. Stimulation at 0.2 Hz and 10 Hz induced respectively single e.j.ps without contractions and summated e.j.ps associated with a contractile response. 3. omega-Conotoxin (0.1 to 3 nM) inhibited markedly and in a concentration-dependent manner both the contractions and e.j.ps to electrical field stimulation. The concentration-response curves to exogenous noradrenaline and KCl remained unaffected. 4. The time-course for the effects of omega-conotoxin (0.3 to 3 nM) indicated a slow onset of action with at least one hour to achieve an equilibrium. 5. The experiments indicate that omega-conotoxin acts prejunctionally to inhibit sympathetic neurotransmission in rat small arteries presumably by inhibition of noradrenaline release. We suggest that omega-conotoxin could be a useful tool to study the control of vascular tone through the autonomic nervous system.

Pharmacological and functional characterization of the guinea-pig B2 bradykinin receptor stably expressed in CHO-K1 cell line.

In the present study, pharmacological properties of a bradykinin B(2) receptor amplified either from guinea-pig ileum or lung and homologous to the previously reported sequence except two amino-acid changes L(124)-->P and N(227)-->Y in the receptor protein were characterized. Tritiated bradykinin ([(3)H]-BK) specifically bound to the cloned guinea-pig B(2) bradykinin receptor stably expressed in Chinese hamster ovary cells (CHO-K1) with a K(D) value of 0.29+/-0.07 nM. In competition experiments, bradykinin (BK) affinity constant value was 0.21+/-0.05 nM while the two specific kinin B(1) ligands, des-Arg(9)-bradykinin (DBK) and des-Arg(9)-Leu(8)-bradykinin (DLBK) were unable to compete with [(3)H]-BK. As the specific peptide antagonist D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-bradykinin (HOE140), (E)-3-(6-acetamido-3-pyridil)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657) and 1-[[3-[2,4-dimethylquinolin-8-yl)oxymethyl] - 2,4 - dichloro - phenyl]sulfonyl] - 2(S) - [[4-[4-(aminoiminomethyl)-phenylcarbonyl]piperazin-1-yl]carbonyl]pyrrolidine (LF16-0335C) exhibited a high affinity for this receptor with K(i) values of 7.34+/-2.45 nM and 8.54+/-1.55 nM respectively. BK and kallidin (KD) increased inositol phosphates (IPs) levels with EC(50) values of 0.44+/-0.12 nM and 6.88+/-0.28 nM, respectively. Neither DLBK nor DBK (0.01 nM to 10 microM) stimulated or inhibited IPs turnover and as expected HOE140 did not raise IPs production. HOE140 (0.1 microM) and LF 16-0335c (1 microM) right shifted the BK response curve with pK(B) values of 9.2+/-0.4 and 8.4+/-0.3, respectively. The results indicate that this cloned guinea-pig receptor displayed typical pharmacological properties of a bradykinin B(2) receptor and support the existence of a single B(2) receptor in this species.

Mediation by 5-HT1D receptors of 5-hydroxytryptamine-induced contractions of rabbit middle and posterior cerebral arteries.

1. 5-Hydroxytryptamine (5-HT) receptor-mediated contraction of endothelium denuded rabbit middle (MCA) and posterior (PCA) cerebral arteries was characterized by use of selective agonists and antagonists for different 5-HT receptor subtypes. 2. 5-HT and various 5-HT receptor agonists contracted the arteries with the following rank order of potency in MCA: 5-carboxamidotryptamine (5-CT) > 5-HT > 5-methoxytryptamine (5-MeOT) > sumatriptan > alpha-methyl-5-HT (alpha-Me-5-HT) >> 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and in PCA: 5-CT > 5-HT > sumatriptan > 5-MeOT > alpha-Me-5-HT >> 8-OH-DPAT. With few exceptions, the maximal contractile responses of these agonists were similar to that induced by 5-HT. 3. The selective antagonists of 5-HT2A/2C (ketanserin), 5-HT4 (SDZ 205-557) and 5-HT1A/1B (S-(-)-propranolol) sites were devoid of inhibitory effect on 5-HT-mediated contraction in both MCA and PCA, thus excluding activation of the corresponding receptors. 4. In both arteries, the contraction-response curve to 5-HT was unaffected by the 5-HT3 receptor antagonist, ICS 205-930 (0.01 and 0.1 microM) whilst a small (3 and 6 fold displacement) was seen with MDL 72222 (0.1 and 1 microM). 5. The mixed 5-HT1-like/5-HT2A receptor antagonist, methiothepin (0.001-0.1 microM), was a potent antagonist of 5-HT-induced contractions in both arteries, giving pA2 values of 9.4 +/- 0.7 and 9.6 +/- 0.8 in MCA and PCA, respectively. 6. Rauwolscine (O.1-10 MicroM) and yohimbine (0.3, 3 MicroM) inhibited contractions to 5-HT in a competitive manner, pA2 values of 7.1 +/- 0.6 and 6.7 +/-0.6 were determined for rauwolscine in MCA and PCA,respectively. An apparent pA2 value of 6.9 +/-0.2 was calculated for yohimbine (3 MicroM) in both MCA and PCA.7. In conclusion, these results suggest that the contractile response to 5-HT in rabbit isolated MCA and PCA is predominantly mediated by the 5-HTID receptor subtype, although a small contribution by 5-HT3 receptors cannot be excluded.

Comparison of contractile responses to 5-hydroxytryptamine and sumatriptan in human isolated coronary artery: synergy with the thromboxane A2-receptor agonist, U46619.

1. The interaction between the thromboxane A2 receptor agonist, U46619 and two 5-hydroxytryptamine (5-HT) receptor agonists, the non-selective, naturally occurring agonist, 5-HT and the selective 5-HT1-like agonist, sumatriptan were studied in human epicardial coronary arteries in vitro. 2. Coronary artery rings (2-4 mm in diameter) were prepared from epicardial arteries from explant hearts of patients undergoing heart transplant (cardiomyopathy, n = 13; ischaemic heart disease, n = 10) and unused donor hearts (n = 5). Each ring of artery was set at optimal resting conditions to record changes in isometric force. 3. The majority of artery rings developed phasic, rhythmic contractions either spontaneously or in response to all vasoconstrictor agonists tested. Both the spontaneous and agonist-induced phasic contractions were abolished by nifedipine (0.1 microM). 4. Concentration-contraction curves to 5-HT-receptor agonists and noradrenaline (NA), were first constructed in artery rings that did not develop phasic activity. 5-HT and ergometrine were the most potent agonists with EC50 values of 6.8 +/- 0.2 and 7.7 +/- 0.2 (-log M) respectively. Potencies (EC50's) to sumatriptan, methysergide and noradrenaline could not be determined due to their poor ability to contract the coronary artery. Maximum contractions (Emax; normalized as a percentage of the contraction to a maximum-depolarizing concentration of K+ in physiological salt solution (KPSS)) for 5-HT, ergometrine, sumatriptan, methysergide and noradrenaline were 40 +/- 10, 9 +/- 3, < 5, < 5 and < 5% respectively. 5. In arteries without phasic activity, U46619 (1 nM) caused an increase in force of 3.8 +/- 1% KPSS. With U46619 present, the Emax values for 5-HT, ergometrine, sumatriptan and methysergide were all markedly increased. For 5-HT and sumatriptan, E., values were 92+/- 4% and 49 +/- 14% KPSSrespectively. The presence of U46619 did not significantly change the sensitivity (EC50) to 5-HT.6. In a separate series of arteries, nifedipine (0.1 microM) was used to block phasic, contractile activity. The synergy observed between U46619 and 5-HT or sumatriptan still occurred although the Emax values for each agonist were depressed but the EC50 values were again unaffected.7. In conclusion, these in vitro studies indicate that the normally poor contractions to sumatriptan, inhuman coronary arteries are significantly enhanced when active force is induced with a thromboxane A2-receptor agonist, U46619. The enhanced response is not specific for either sumatriptan or 5-HT,-like receptors since contractions to 5-HT, ergometrine and methysergide were also potentiated by U46619.

Induction of kinin B1 receptor-dependent vasoconstriction following balloon catheter injury to the rabbit carotid artery.

1. Balloon catheter injury to the rabbit carotid artery damaged the endothelium and induced neointima formation over 7 days. The area of intima, expressed as a percentage of the media, was 16.2 +/- 4.2% and 8.2 +/- 0.1% in balloon catheter-injured and sham-operated arteries. 2. Seven days after arterial injury, carotid arteries were isolated and set up as ring preparations in organ baths for isometric tension measurements. Balloon catheter-injured arteries first contracted with noradrenaline (0.01-0.1 microM), contracted further in a concentration-dependent manner to bradykinin (BK; pD2, 5.98 +/- 0.22; Emax, 41.3 +/- 5.2% of KCl) and to des-Arg9-BK (pD2, 7.12 +/- 0.36; Emax, 46.0 +/- 9.9% of KCl). In contrast, vessel segments with endothelium either intact or acutely removed were unresponsive to both BK receptor agonists. 3. The concentration-contraction curves for BK and for des-Arg9-BK were shifted to the right by the B1 receptor antagonist, [Leu8]des-Arg9-BK (3 microM), but not by the selective B2 receptor antagonist, Hoe 140 (1 microM). 4. Thus, BK and its metabolite, des-Arg9-BK act as vasoconstrictor agents following balloon catheter injury. These effects appear to be mediated by activation of B1 receptors.

Stable expression of human kinin B1 receptor in 293 cells: pharmacological and functional characterization.

1. We compared the binding properties of [3H]-desArg10-[Leu9]-kallidin, a radiolabelled kinin B1 receptor antagonist, to membranes from IMR-90 human embryonic fibroblasts and from 293 cells transiently or stably transfected with the human B1 receptor. 2. The dissociation constant (KD) of [3H]-desArg10-[Leu9]-kallidin and the affinity of several kinin receptor agonists and antagonists were similar between the native and cloned receptor, either transiently or stably expressed in 293 cells. In IMR-90 cells, the rank order of potency was that expected for a kinin B1 receptor. 3. The receptors transiently or stably expressed in 293 cells were fully functional with respect to their signalling properties. Phosphoinositide hydrolysis was increased in a concentration-dependent manner by the B1 receptor agonist, desArg10-kallidin. Functional coupling to the calcium pathway was also demonstrated for the native and stably expressed human B1 receptor. 4. In conclusion, the established stable and functional 293 cell clone may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of the kinin B1 receptor.

The kinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin: an antagonist of the angiotensin AT1 receptor which also binds to the AT2 receptor.

1. Agonists and antagonists of kinin B1 and B2 receptors were evaluated in vitro for their effects against angiotensin II (AII)-induced contractile responses in the rabbit aorta and for their binding properties to angiotensin AT1 and AT2 receptors from purified membrane of rat liver and lamb uterus respectively. 2. In aortic rings, the kinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin (BK) (3-100 microM) caused a concentration-dependent decrease in sensitivity and a depression of the maximum response to AII. Des-Arg10-[Leu9]kallidin (KD), des-Arg9-BK, des-Arg10-KD, BK or KD at 3 microM had no effect against AII-induced contractions. 3. Des-Arg9-[Leu8]BK (3 or 100 microM) did not affect contractions of aortic rings to histamine, potassium chloride, endothelin-1, 5-hydroxytryptamine, noradrenaline and the thromboxane A2-mimetic, U46619. 4. Des-Arg9-[Leu8]BK displaced [125I]-Sar1-AII binding to the AT1 subtype in rat liver membranes with a Ki value of 1.1 +/- 0.4 microM. Values of Ki for des-Arg9-BK and KD were 45 +/- 13 microM and 25 +/- 22 microM, respectively. The other kinin derivatives des-Arg10-KD, BK and des-Arg10-[Leu9]KD at concentrations up to 100 microM did not bind to the AT1 receptor. 5. All the kinin derivatives except BK bound to AT2 receptors in lamb uterus membranes. Values of Ki for des-Arg9-[Leu8]BK, des-Arg10-[Leu9]KD, des-Arg9-BK, des-Arg 10-KD and KD were 0.3 +/- 0.1, 0.7 +/- 0.1, 1.2 +/- 0.3, 1.5 +/- 0.3 and 7.0 +/- 1.6 microM, respectively. 6. In conclusion, des-Arg9-[Leu8]BK is an insurmountable antagonist of AII-induced contractions in the rabbit aorta and also binds with a relatively high affinity to AT1 and AT2 receptors in isolated membrane fractions. These additional properties of des-Arg9-[Leu8]BK should be considered when it is used as an antagonist to characterize kinin B1 receptors.

Pharmacological evidence for a single bradykinin B2 receptor in the guinea-pig.

1. The present study addresses the possibility of the existence of different kinin B2 receptor subtypes in the guinea-pig by evaluating the affinity of peptide and nonpeptide receptor antagonists. For this purpose, jugular vein rings, ileum segments, lung parenchymal and trachea strips were set up in organ baths for isometric tension measurements. The experiments were conducted in the presence o indomethacin (3 microM), atropine (10 microM) and captopril (10 microM). 2. BK contracted jugular vein (JV), ileum (GPI), parenchyma (LP) and trachea (GPT) with an EC50 of 13.2 +/- 1.4 nM (n=27), 11.2 +/- 2.1 (n=26), 23.6 +/- 6.3 (n=26), and 33.0 +/- 6.5 (n=27), respectively. Thiorphan, a neutral endopeptidase (EC 3.4.34.11) inhibitor and MERGETPA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), a carboxypeptidase inhibitor, had no effect on the BK-induced contractions of JV, GPI and LP. In the GPT, thiorpan potentiated the contractile response to BK and was thus added in the corresponding experiments. 3. The peptide B2 receptor antagonist, Hoe 140 and the nonpeptide compound, WIN 64338, behaved as noncompetitive antagonists against contractile responses to cumulative BK in the four tissues although Hoe 140 appeared as a competitive inhibitor in the GPT only. IN order to compare the inhibitory potency of these compounds between tissues, pKB values were determined. Mean values of pKB for Hoe 140 were 8.05 +/- 0.07, 8.43 +/- 0.11, 8.13 +/- 0.18, and 8.52 +/- 0. 25 in the JV, GPI, GPT and LP, respectively. WIN 64338 gave mean pKB values of 6.89 +/- 0.10, 7.57 +/- 0.12, 7.36 +/- 0.12 adn 7.51 +/- 0.28 in the JV, GPI, LP and GPT, respectively. 4. D-Arg [Hyp3, D-Phe7, Leu8]BK and D-Arg[Hyp3, D-Phe7]BK (NPC 567) inhibited in a competitive fashion the concentration-response curves to BK. Values of pA2for each compound were not significantly different in the four tissues and were between 5.81 and 6.31 for D-Arg [Hyp3, D-Phe7, Leu8]BK and between 5.55 and 5.65 for NPC 567.

Haemodynamic and cardiac effects of kinin B1 and B2 receptor stimulation in conscious instrumented dogs.

1. Mongrel dogs were chronically instrumented with an intra-aortic catheter, a Königsberg intraventricular pressure transducer and a Döppler flow probe around the left coronary artery. After ganglionic blockade with hexamethonium, the cardiovascular effects of bradykinin B1 and B2 receptor agonists, des-Arg9-bradykinin and bradykinin (BK), were investigated in the presence and absence of specific antagonists. The contribution of nitric oxide (NO) and prostanoids to the cardiovascular effects of kinins was also examined. 2. BK (1 microgram kg-1 min-1) and des-Arg9-BK (1 microgram kg-1 min-1) both given as a 2 min i.v. infusion, produced a significant decrease in mean arterial pressure (MAP, -34 +/- 4% for BK and -45 +/- 2% for des-Arg9-BK) and coronary vascular resistance (CVR, -37 +/- 5% for BK and -50 +/- 2% for des-Arg9-BK), without affecting cardiac contractility, left ventricular end diastolic pressure, and coronary velocity. BK caused a significantly greater decrease in MAP and CVR than des-Arg9-BK (P < 0.05). 3. Pretreatment with the B1 receptor antagonist, des-Arg9-[Leu8]-BK (25 micrograms kg-1) significantly inhibited the decrease in MAP and CVR produced by des-Arg9-BK but not by BK. Infusion of des-Arg9-[Leu8]-BK alone also induced a significant decrease in MAP and CVR (P < 0.05). In the presence of the B2 receptor antagonist, Hoe 140 (25 micrograms kg-1), only the decreases in MAP and CVR caused by BK were significantly reduced (P < 0.05). 4. Inhibition of NO synthase with N omega-nitro-L-arginine (L-NOARG, 45 mg kg-1) significantly (P < 0.05) prevented the decrease in CVR but not MAP induced by des-Arg9-BK, whilst responses to BK were not affected by L-NOARG pretreatment. Inhibition of prostanoid synthesis with indomethacin (25 mg kg-1) did not affect the reductions in MAP and CVR induced by des-Arg9-BK or BK. 5. In conclusion, i.v. des-Arg9-BK and BK administration induced reductions in MAP and CVR suggesting that in conscious instrumented dogs both B1 and B2 receptors are present and can affect systemic blood pressure and coronary resistance regulation. Our results also suggest that prostanoids are not involved in the vascular response to kinins and that coronary vascular B1 receptors are at least in part coupled to the release of NO.

LF 16.0335, a novel potent and selective nonpeptide antagonist of the human bradykinin B2 receptor.

1. In the present paper, we describe the in vitro pharmacological properties of LF 16.0335 (1-[[3-[(2,4-dimethylquinolin-8-yl)oxymethyl]-2,4-dichloro-p henyl]sulphonyl] -2(S) - [[4 -[4-(aminoiminomethyl)phenylcarbonyl]piperazin-1-yl]ca rbonyl]pyrrolidine), a novel and potent nonpeptide antagonist of the human bradykinin (BK) B2 receptor. 2. LF 16.0335 displaced [3H]-BK binding to membrane preparations from CHO cells expressing the cloned human B2 receptor, INT 407 cells and human umbilical vein with Ki values of 0.84+/-0.39 nM, 1.26+/-0.68 nM and 2.34+/-0.36 nM, respectively. 3. In saturation binding studies performed in INT 407 cell membranes in the presence or absence of LF 16.0335, max values of [3H]-BK were not significantly changed suggesting that LF 16.0335 behaves as a competitive antagonist. 4. LF 16.0335 had no affinity for the cloned human kinin B1 receptor stably expressed in 293 cells. In addition, this compound at 1 microM did not significantly bind to a range of 40 different membrane receptors and eight ion channels except muscarinic M2 and M1 receptors for which an IC50 value of 0.9 and 1 microM was obtained. 5. BK stimulates in a concentration-dependent manner phosphoinositosides (IPs) production in cultured INT 407 cells. Concentration-response-curves to BK were shifted to the right in the presence of LF 16.0335 (0.1 microM) without reduction of the maximum. LF 16.0335 inhibited the concentration-contraction curve to BK in the human umbilical vein giving a pA2 value of 8.30+/-0.30 with a Schild plot slope that was not different from unity. 6. These results demonstrate that LF 16.0335 is a potent, selective and competitive antagonist of the human bradykinin B2 receptor.

Pharmacological and molecular evidence for kinin B1 receptor expression in urinary bladder of cyclophosphamide-treated rats.

1. In the present study, we developed an experimental model of cystitis induced by cyclophosphamide (CYP). In order to characterize des-Arg9-BK-induced contraction on the urinary bladder (UB) during the development of inflammation and to quantify kinin B1 receptor gene expression using a quantitative RT - PCR technique. 2. In the presence of peptidase inhibitors captopril (10 microM), DL-thiorphan (1 microM) and DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MERGEPTA 5 microM), bradykinin (BK) (0.3 - 3,000 nM) evoked a concentration-dependent contraction of rat UB which was not different between the CYP- and vehicle-treated groups. Unlike BK, des-Arg9-BK (0.3 - 100,000 nM) did not contract UB from vehicle-treated rats but contracted vigorously bladder strips from CYP-treated rats 14, 24 and 168 h after treatment. In UB of 24 h treated rat, the pD2 value of des-Arg9-BK was 7.3+/-0.1. 3. The cyclo-oxygenase inhibitor indomethacin (3 microM) reduced by 30% the maximal response of des-Arg9-BK. Both the kinin B1 receptor antagonists des-Arg9-[Leu8]BK (10 microM) and des-Arg10-Hoe 140 (10 microM) produced a rightward shift of the concentration-response curve to des-Arg9-BK yielding pKB values of 6.8+/-0.2 and 7.2+/-0.1, respectively, whilst the kinin B2 receptor antagonist Hoe 140 (1 microM) had no effect. 4. After CYP treatment, mRNA coding for the kinin B1 receptor appeared predominantly in UB. In this organ, the induction was progressive, reaching a maximum 48 h after CYP treatment. 5. In conclusion, the present study provides strong evidence for an induction of kinin B1 receptors in UB of CYP-treated rats. This was associated at a molecular level with an increase in mRNA expression of the gene coding for the kinin B1 receptor. This kinin receptor displayed the whole features of a classical rat kinin B1 receptor.

Pharmacological characterization of the bradykinin B2 receptor: inter-species variability and dissociation between binding and functional responses.

1. The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1- oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinoli nyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. 2. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8-6.6 fold and 7.0-16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6-23 fold in physiological HBSS compared to low ionic strength TES binding buffer. 3. BK (0.01-3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10(-7) M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. 4. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). 5. FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and native bradykinin B2 receptor in human (pKi of 8.66 and 8.59, respectively) and in rat (pKi 9.67 and 9.81, respectively). 6. In conclusion, we suggest that the binding buffer composition has to be taken into account when screening new compounds and that inter-species differences should be considered when setting up animal models with the aim of developing bradykinin B2 receptor antagonists as therapeutic agents.