Paget disease of bone: mapping of two loci at 5q35-qter and 5q31.

Paget disease of bone is characterized by focal increases of the bone-remodeling process. It is the second most common metabolic bone disease after osteoporosis. Genetic factors play a major role in the etiology of Paget disease of bone, and two loci have been mapped for the disorder: PDB1 and PDB2. The gene(s) causing the typical form of the disorder remains to be characterized. To decipher the molecular basis of Paget disease of bone, we performed genetic linkage analysis in 24 large French Canadian families (479 individuals) in which the disorder was segregating as an autosomal dominant trait. After exclusion of PDB2, a genomewide scan was performed on the three most informative family nuclei. LOD scores >1.0 were observed at seven locations. The 24 families were then used to detect strong evidence for linkage to chromosome 5q35-qter. Under heterogeneity, a maximum LOD score of 8.58 was obtained at D5S2073, at straight theta= .1. The same characteristic haplotype was carried by all patients in eight families, suggesting a founder effect. A recombination event in a key family confined the disease region within a 6-cM interval between D5S469 and the telomere. The 16 other families, with very low conditional probability of linkage to 5q35-qter, were further used, to map a second locus at 5q31. Under heterogeneity, a maximum LOD score of 3.70 was detected at D5S500 with straight theta=.00. Recombination events refined the 5q31 region within 12.2 cM, between D5S642 and D5S1972. These observations demonstrate the mapping of two novel loci for Paget disease of bone and provide further evidence for genetic heterogeneity of this highly prevalent disorder. It is proposed that the 5q35-qter and 5q31 loci be named "PDB3" and "PDB4," respectively.

Mechanism of the acute pressor effect and bradycardia elicited by diaspirin crosslinked hemoglobin in anesthetized rats.

Diaspirin crosslinked hemoglobin (DCLHb) is a chemically stabilized hemoglobin (Hb) that induces an increase in blood pressure and a decrease of heart rate when injected intravenously in some animals. The mechanism by which DCLHb elicits these hemodynamic effects was studied in pentobarbital-anesthetized, vagotomized rats using a variety of drugs known for their inhibitory action towards endogenous hemodynamically active systems. The hypertensive episode elicited by DCLHb (100 or 400 mg.kg-1) was attenuated in animals pretreated with NG-nitro-L-arginine (inhibitor of nitric oxide synthases) throughout the 30-min period of observation, but it was not reduced in those pretreated with a variety of sympatholytic drugs (e.g., prazosin), atropine, BIBP-3226 (neuropeptide Y antagonist), indomethacin, [1-(beta-mercapto-beta,beta-cyclopentanemethylene propionic acid), 2-(0-methyl) tyrosine]-Arg8 vasopressin (vasopressin antagonist), losartan (angiotensin antagonist), bosentan (endothelin antagonist), or L-arginine-(nitric oxide precursor), compared with control animals. With the exception of propranolol and BIBP-3226, none of the aforenamed inhibitors reduced the amplitude of the bradycardia associated with the pressor effect of DCLHb. These results suggest that: (i) the acute (< 30 min) pressor activity of DCLHb in our animal model requires the presence of an endogenous nitric oxide generating system to be expressed; (ii) the bradycardia elicited by DCLHb might involve the participation of neuropeptide Y and (or) its NPY-1 receptors, but it is unlikely to involve a baroreceptor-mediated vagal reflex, at least in our animal model.

Structural requirements and mechanism of the pressor activity of Leu-Val-Val-hemorphin-7, a fragment of hemoglobin beta-chain in rats.

A rat blood pressure assay was used to perform a structure-activity relationship study (SAR) of Leu-Val-Val-hemorphin-7 (LVV-H7), a fragment of hemoglobin (Hb) beta-chain, elucidate the mechanisms of its cardiovascular effects, and test its potential involvement in the pressor activity of diaspirin crosslinked Hb (DCLHb), a recently developed Hb-based oxygen carrier. The SAR study revealed that the C-terminal-Arg-Phe-amino acid sequence of LVV-H7 contained the main determinants of the pressor activity of this peptide. Drug interaction studies using various inhibitory drugs (e.g., phentolamine, clonidine, etc.) and LVV-H7 showed that the pressor effect and tachycardia elicited by LVV-H7 involved the activation of the sympathetic nervous system (SNS). Additional studies using phenytoin (sodium channel blocker), [Tic7]H7(5-7)-NH2 (putative antagonist of receptors for LVV-H7) and H7(5-7)-NH2, an amidated C-terminal fragment of LVV-H7, suggested that LVV-H7 activated the SNS by interacting with specific receptors functionally coupled with phenytoin-sensitive sodium channels. The pressor effect and tachycardia caused by LVV-H7 were potentiated by captopril, suggesting that the angiotensin converting enzyme may contribute to the inactivation of LVV-H7 in rats. The pressor activity of DCLHb, in contrast to that elicited by LVV-H7, was not affected by animal pretreatment with LVV-H7 fragments shown to inhibit the pressor effect of LVV-H7. We conclude that: 1) LVV-H7 is unlikely to mediate the pressor activity of DCLHb in rats; 2) the pressor and tachycardic activities of LVV-H7 are mediated by the SNS; 3) the C-terminal-Arg-Phe-amino acid sequence of LVV-H7 contains the chemical groups responsible for the pressor effect of this peptide in rats; 4) LVV-H7 and FMRF amide-related peptides may share the same mechanism of pressor activity in rats.

Nimodipine inhibits the pressor activity of diaspirin-crosslinked hemoglobin (DCLHb) in the rat.

Impaired nitric oxide (NO) activity is associated with an increase in blood pressure in rats. Voltage-regulated calcium channels are believed to participate in this hemodynamic event. To further test this hypothesis, we examined the effect of nimodipine and verapamil (calcium antagonists) on the pressor activity of diaspirin-crosslinked hemoglobin (DCLHb), a well-known NO scavenger, in anesthetized rats. Nimodipine, the most potent of the two calcium antagonists used, was also tested against phenylephrine (alpha1-adrenoceptor agonist). The pressor effect of DCLHb was reduced markedly by nimodipine and verapamil, whereas that elicited by phenylephrine, particularly the tonic phase of its pressor response, was resistant to blockade by nimodipine. The bradycardia and tachycardia associated with the pressor effects of DCLHb and phenylephrine, respectively, were not affected by nimodipine. The pressor effect elicited by DCLHb and its alteration by nimodipine were also examined in rats pretreated with 100% O2. This treatment was found to potentiate the pressor effect of DCLHb. However, this synergism did not impair the inhibitory action of nimodipine towards the pressor activity of DCLHb. Altogether these results suggest that the pressor activity of DCLHb in our animal model might involve the participation of voltage-regulated calcium channels.

Contribution of angiotensin-converting enzyme to the cardiac metabolism of bradykinin: an interspecies study.

The role of angiotensin-converting enzyme (ACE) in the metabolism of bradykinin (BK) has been studied in several tissues. However, and contrary to angiotensin I, the metabolism of BK at the cardiac level has not been investigated. In this study, we define the participation of ACE in the carboxy-terminal degradation of BK in heart membranes of the dog, human, rabbit, and rat. The calculation of the kinetic parameters characterizing the metabolism of BK and the generated des-Arg9-BK can be summarized as follows: the half-life (t1/2) of BK [dog (218 +/- 32 s) > human (143 +/- 9 s) = rat (150 +/- 4 s) > rabbit (22 +/- 2 s)] and of des-Arg9-BK [dog (1,042 +/- 40 s) > human (891 +/- 87 s) > rat (621 +/- 65 s) > rabbit (89 +/- 8 s)] both showed significant differences according to species. Enalaprilat, an ACE inhibitor, significantly prevented the rapid degradation of BK and des-Arg9-BK in all species studied, whereas retrothiorphan, a neutral endopeptidase inhibitor, and losartan, an angiotensin II type I receptor antagonist, did not affect this metabolism. The relative importance of ACE in the cardiac metabolism of BK was species related: dog (68.4 +/- 3.2%) = human (72.2 +/- 2.0%) > rabbit (47.7 +/- 5.0%) = rat (45.3 +/- 3.9%). ACE participation in the metabolism of des-Arg9-BK was as follows: rabbit (57.0 +/- 4.0%) > dog (39.9 +/- 8.8%) = human (25.4 +/- 5.5%) = rat (36.0 +/- 7.0%). The participation of cardiac kininase I (carboxypeptidase M) in the transformation of BK into des-Arg9-BK was minor: human (2.6 +/- 0.1%) > dog (0.9 +/- 0.1%) = rabbit (1.0 +/- 0.1%) = rat (1.0 +/- 0.1%). These results demonstrate that ACE is the major BK-degrading enzyme in cardiac membranes. However, the metabolism of exogenous BK by heart membranes is species dependent. Our observations could explain some discrepancies regarding the contribution of kinins in the cardioprotective effects of ACE inhibitors.

Involvement of endogenous kinins in the pathogenesis of peptidoglycan-induced arthritis in the Lewis rat.

OBJECTIVE: To investigate the pathophysiologic roles of endogenous bradykinin (BK) and des-Arg9-BK on local and systemic inflammatory responses in a rat model of acute arthritis induced by peptidoglycan-polysaccharide (PG-APS). METHODS: Female Lewis rats were injected intraperitoneally with PG-APS. Selective antagonists of B1 (Lys-[Leu8]-des-Arg9-BK) and B2 (Hoe 140) receptors were infused at 500 microg/kg and 5 mg/kg per day for 6 days, starting 3 days before induction of inflammation, with subcutaneous micro-osmotic pumps. The local inflammatory response was assessed by paw edema, joint swelling, and tissue content of BK and des-Arg9-BK. These peptides were measured by highly sensitive and specific chemiluminescent enzyme immunoassays. Systemic inflammatory reaction was evaluated by the hepatic concentration of the type 2 acute-phase protein T-kininogen. RESULTS: PG-APS induced significant paw edema and joint swelling 24-72 hours after intraperitoneal injection. The maximal responses to PG-APS observed at 72 hours were significantly reduced (31-38%) by the combination of both B1 and B2 receptor antagonists at 5 mg/kg per day. PG-APS induced a significant increase of BK (up to 5.3-fold) and des-Arg9-BK (up to 4.1-fold) 72 hours after challenge. Liver T-kininogen content was increased by 5.3-, 7.7-, and 5.8-fold at 24, 48, and 72 hours, respectively, after PG-APS injection. At 24 hours, Hoe 140 and Lys-[Leu8]-des-Arg9-BK increased liver T-kininogen content by 43% and 45%, respectively, but they had no effect at 72 hours. CONCLUSION: The results indicate that endogenous kinins are involved in local and systemic acute inflammatory responses, through both B1 and B2 kinin receptors, in the model of PG-APS-induced arthritis.

Cardiovascular effects of Sar-[D-Phe8]des-Arg9-bradykinin, a metabolically protected agonist of B1 receptor for kinins, in the anesthetized rabbit pretreated with a sublethal dose of bacterial lipopolysaccharide.

We investigated the mechanism of the hypotensive effect of Sar-[D-Phe8]des-Arg9-bradykinin (BK) in lipopolysaccharide-treated anesthetized rabbits. The study involved pharmacokinetic and hemodynamic measurements and tests of antagonism with various drugs. The rate of elimination of Sar-[D-Phe8]des-Arg9-BK from the rabbit plasma was slower than that of Lys-BK, a naturally occurring B1 agonist. The amplitude of the hypotensive effect of Sar-[D-Phe8]des-Arg9-BK was not affected by pretreatment with indomethacin, diclofenac, dazmegrel, NG-nitro-L-arginine, glibenclamide, MK-886, BN-50739, atropine or propranolol, but its duration was shortened by indomethacin and diclofenac. Sar-[D-Phe8]des-Arg9-BK-induced hypotension was associated with decreases of total peripheral resistance, cardiac output, carotid, mesenteric and femoral blood flow, transient reductions followed by secondary increases of vascular resistance in the carotid and femoral beds, reductions of central venous pressure, but no change of hematocrit. Animal pretreatment with diclofenac or hexamethonium abolished the secondary increases of carotid bed vascular resistance caused by the B1 agonist. These and other results suggest that peripheral vasodilation leading to a decrease of total peripheral resistance and a decrease of cardiac output may both contribute consecutively to the hypotensive effect of Sar-[D-Phe8]des-Arg9-BK in this animal model. Inappropriate compensatory responses to arterial hypotension, prostaglandin release, and slow rate of elimination of Sar-[D-Phe8]des-Arg9-BK from the rabbit plasma, may all be at the basis of the prolonged duration of the hypotension caused by the B1 agonist.

Digoxigenin-labeled peptides for the immunological quantification of intracellular signaling proteins: application to the MAP kinase kinase isoform MEK2.

Two competitive enzyme immunoassays using digoxigenin-labeled peptides have been developed for the quantification of the protein kinase MEK2 in cell extracts. Rabbit polyclonal antibodies directed against either the amino-terminal or proline-rich amino acid sequences of MEK2 were used for the immunoconcentration of the protein. Anti-digoxigenin Fab fragments labeled with horseradish peroxidase allowed the detection of the immune complexes. Amino-terminal and proline-rich enzyme immunoassays exhibited a sensitivity level of 63 and 71 fmol/mL, respectively, and displayed a half-maximal saturation value of 1320 and 1780 fmol/mL. The intra- and inter-assay coefficients of variation for both assays assessed at three different concentrations of MEK2 were lower than 6% and 12%, respectively. The amount of MEK2 measured by the two methods demonstrated an excellent correlation with the expression level of the protein detected by immunoblot analyses when tested on different cell lysates.

Mechanism of the contractile effect of diaspirin-cross-linked hemoglobin in rat isolated aorta strip denuded of endothelium as revealed using an oil-immersion procedure.

Diaspirin-cross-linked hemoglobin (DCLHb) is a chemically modified hemoglobin (Hb) (i.e., alpha-subunits are cross-linked by a covalent bond) currently being tested as a potential oxygen-carrying blood substitute. It was examined for possible vasoactive properties, using the rat isolated aorta strip denuded of endothelium. In this experimental model, DCLHb (1.6-155 microM) was found to be inactive as a vasoconstrictor when added to the Krebs medium but to elicit contractile responses once the Krebs medium containing DCLHb was replaced by mineral oil, a procedure that favors the sequestration of a fixed amount of DCLHb within a substantially reduced volume of extracellular fluid. The contractile activity of DCLHb in our experimental model (i.e., prior exposure of tissues to drugs in the Krebs medium followed by replacement of the Krebs medium by mineral oil) was mimicked by methemoglobin and metmyoglobin, but not by cytochrome c, albumin, hemin, hematin, Fe2+, and a variety of hemorphins. It was abolished by indomethacin, SQ-29548 (prostaglandin H2-thromboxane A2 receptor antagonist), thiourea, or N-2-mercaptopropionylglycine (MCPG), reduced partially by verapamil, but not affected by dazmegrel, MK-886 (leukotriene biosynthesis inhibitor), dimethylsulfoxide, vitamin C or E, deferoxamine, NG-nitro-L-arginine, naloxone, and a variety of other drug receptor antagonists (e.g., prazosin) and protease inhibitors (e.g., pepstatin). Rat aorta strips denuded of endothelium exhibited contractile responses to arachidonic acid added in the Krebs medium (i.e., with no mineral oil added afterwards). Such contractile activity was reduced by SQ-29548, thiourea, or MCPG. Addition of U-46619 (prostaglandin H2-thromboxane A2 mimetic) to the Krebs medium also elicited contractile responses in rat aorta strips denuded of endothelium. Such contractile activity was reduced by SQ-29548, thiourea, or verapamil but not by MCPG. Within the limitations of our experimental approach, these results suggest that (1) the contractile activity of DCLHb in rat aorta strips denuded of endothelium following replacement of the Krebs medium by mineral oil involves the participation of a secondary mediator, which could be a vasoconstrictor metabolite of arachidonic acid; (2) the participation of reactive oxygen species, potential degradation products of DCLHb (e.g., heme, Fe2+, hemorphins), or other mediators in the contractile activity of DCLHb is unlikely; and (3) Ca2+ entry into target cells might be involved in the process by which DCLHb elicits its contractile activity in our experimental model.

Kinin-induced prolongation of action-potential duration in right ventricular muscle from rat: involvement of B1 and B2 receptors.

Previous work has shown that, in rat ventricular muscle, bradykinin (BK) causes a dose-dependent increase in action potential duration (APD), an action that may be responsible for APD prolongation by captopril (kininase II). To determine which kinin receptor might be involved in APD prolongation, we studied the effects of B1- and B2-receptor agonists, as well as those of antagonists and mergepta (a kininase I inhibitor) added during BK superfusion. Action potentials were recorded by using the standard glass microelectrode technique in rat ventricular muscle preparations. Action-potential characteristics were compared between preparations superfused with peptide/drug-free Tyrode's solution (control group) and preparations superfused with peptide/drug-containing solution. APD was significantly longer in preparations superfused with BK (10(-8) M) than in the control group. The APD prolongation induced by BK, a known B2-receptor agonist, was significantly reduced by Hoe 140 (a B2 antagonist) and also by Lys[Leu8]des-Arg9-BK (a B1 antagonist), an action presumably related to inhibition of B1 receptor stimulation by the BK metabolite des-Arg9-BK. When mergepta was added in the presence of BK, APD prolongation by BK was significantly reduced, an effect that could have been related to reduced B1-receptor stimulation after inhibition of the endogenous generation of des-Arg9-BK by kininase I. Sar4-[d-Phe8]des-Arg9-BK, a B1-receptor agonist that is not degraded by kininase II, also prolonged APD. We conclude that both B1 and B2 receptors may be involved in APD prolongation induced in rat ventricular muscle preparations.

Bradykinin decreases T-kininogen synthesis in a rat hepatoma cell line: evidence of bradykinin B2-type receptors.

Using the rat H4-II-E-C3 hepatoma cell line, we investigated the presence of [125I][Tyr8]BK binding sites and the direct modulation of T-kininogen synthesis, an acute phase protein of inflammation, by bradykinin (BK) analogues. H4-II-E-C3 membrane preparations exhibited [125I][Tyr8]BK binding sites with a Kd of 4 nM and a Bmax of 120 fmol/mg of protein. Des-Arg9-BK showed no affinity (Ki > 10(-4) M) for these sites. The B2 metabolism-resistant and selective agonist [Phe8 psi (CH2-NH)Arg9]BK decreased the T-kininogen concentration in H4-II-E-C3 medium by 23% (p < 0.05). This effect was reversed by coincubation with the B2 antagonist HOE140. The B1 agonist Sar[D-Phe8]des-Arg9-BK and the B1 antagonist Lys[Leu8]des-Arg9-BK did not modify T-kininogen concentrations. The interaction between cytokines and kinins in the modulation of T-kininogen synthesis was also studied. Preincubation of hepatoma cells for 1 h with interleukin-1 alpha (IL-1 alpha) alone reduced T-kininogen concentrations by 37%, and this effect was blocked by co-addition of HOE140. Preincubation with interleukin-6 (IL-6) increased T-kininogen levels by threefold. Coincubation in the presence of the B2 agonist decreased this augmentation by 24%. The latter effect was reversed by co-addition of HOE140. None of the cytokines tested induced a response to the B1 agonist or antagonist under the experimental conditions studied. Overall, these results support the presence of a functional B2 receptor on H4-II-E-C3 cells that modulates T-kininogen synthesis. We suggest that the receptor is involved in vivo in a retroaction loop between kinins and T-kininogen production during inflammation. We speculate that BK could be a mediator in the modulation of acute phase protein synthesis by the cytokines IL-1 alpha and IL-6.

Cloning and pharmacological characterization of the rabbit bradykinin B2 receptor.

Degenerate primers, corresponding to consensus sequences of third and sixth transmembrane domains of G protein-coupled receptor superfamily, were used for the polymerase chain reaction amplification and consecutive characterization of G protein-coupled receptors present in cultured rabbit aortic smooth muscle cells. One of the isolated resulting fragments was highly homologous to the corresponding region of the bradykinin (BK) B2 receptor cloned in other species. The polymerase chain reaction fragment was used to screen a rabbit genomic library, which allowed the identification of an intronless 1101-nucleotide open reading frame which codes for a 367-amino acid receptor protein. The rabbit B2 receptor sequence is more than 80% identical to the ones determined in three other species and retain putative glycosylation, palmitoylation and phosphorylation sites. In the rabbit genomic sequence, an acceptor splice sequence was found 8 base pairs upstream of the start codon. Northern blot analysis showed a high expression of a major transcript (4.2 kilobases) in the rabbit kidney and duodenum, and a less abundant expression in other tissues. Southern blot experiments suggest that a single copy of this gene exists in the rabbit genome. The cloned rabbit B2 receptor expressed in COS-1 cells binds [3H]BK in a saturable manner (KD 2.1 nM) and this ligand competes with a series of kinin agonists and antagonist with a rank order consistent with the B2 receptor identity. The insurmountable character of the antagonism exerted by Hoe 140 against BK on the rabbit B2 receptor, previously shown in pharmacological experiments, was confirmed in binding experiments with the cloned receptor expressed in a controlled manner. By contrast, Hoe 140 competed with [3H]BK in a surmountable manner for the human B2 receptor expressed in COS-1 cells. The cloning of the rabbit B2 receptor will be useful notably for the study of the structural basis of antagonist binding and for studies on receptor regulation in a relatively large animal.

Regulation of kinin-induced contraction and DNA synthesis by inflammatory cytokines in the smooth muscle of the rabbit aorta.

1. In rabbit aortic rings, the contractile response to kinins is mediated by the B1 receptors for kinins; the response is upregulated from an initial null level in a time- and protein synthesis-dependent manner. Incubation (3 h) with human recombinant interleukin-1 beta (IL-1 beta) selectively amplified the contractile response to the B1 receptor agonist Sar-[D-Phe8]des-Arg9-BK, while it did not affect the contractile effect of other agents (angiotensin II, endothelin-1, phenylephrine). 2. Oncostatin M (OSM), but not macrophage migration inhibitory factor (MIF), increased the contractile response to the B1 receptor agonist, des-Arg9-bradykinin (des-Arg9-BK). 3. Cultured smooth muscle cells derived from the rabbit aorta exhibit a significant des-Arg9-BK-induced increase in [3H]-thymidine incorporation if pretreated with a cyclo-oxygenase inhibitor (diclofenac) and concomitantly treated with the cytokines IL-1 or OSM. Angiotensin II, endothelin-1 or phenylephrine, alone or in the presence of IL-1 beta, exerted little effect on DNA synthesis in these cells. 4. The pharmacological characterization of the mitogenic response to kinins using a set of agonist and antagonist analogues is consistent with mediation by B1 receptors. Des-Arg9-BK-induced DNA synthesis is suppressed by prostaglandin E2 by a prostacyclin mimetic (iloprost), by the Ser/Thr protein kinase inhibitor, H-7, and by a tyrosine kinase inhibitor (i.e. an erbstatin analogue). 5. B1 receptor-mediated responses and their capacity to be regulated by cytokines, are retained in rabbit aortic smooth muscle cells. Such responses could be relevant to tissue repair mechanisms and hypertrophic medial responses to injury in arteries.

Mode of action of thrombin in the rabbit aorta.

1. Thrombin is a vasoactive protease that elicits the contraction of the rabbit aorta by activating a G-protein coupled receptor through cleavage of its N-terminal extracellular domain. Synthetic peptides corresponding to the newly exposed N-terminus, following thrombin cleavage, have been shown to reproduce some of the activities of thrombin in the rabbit aorta. 2. Intracellular pathways involved in the contractile response of the rabbit aorta to thrombin and synthetic peptides were examined by use of a series of inhibitors. A similar method was applied to characterize the mitogenic effect of thrombin on cultured smooth muscle cells (SMCs) derived from the same tissue. 3. Results from this study indicate that the contractile response of the rabbit aorta to thrombin is dependent on the activation of protein kinase C (PKC) and independent of extracellular calcium. The contractile response to thrombin can be fully reproduced by peptide agonists related to the N-terminal receptor sequence. However, subtle differences seem to exist between the mechanism of the contractile effect of thrombin and of the synthetic peptides, as both PKC activation and extracellular calcium were found to participate in the contractile effect of the synthetic peptides. 4. In cultured SMCs, both thrombin and the synthetic peptides increased inositol phosphate turnover; however, only thrombin elicited a mitogenic effect, which occurs at thrombin concentrations well below those needed to increase inositol phosphate turnover significantly. Activation of a tyrosine kinase pathway is involved in the mitogenic effect of thrombin on aortic SMCs. 5. Altogether these results suggest the existence of subtle differences between the mode of action of thrombin and of synthetic peptides related to the N-terminal thrombin receptor sequence, in the rabbit aorta.

Recombinant human hemoglobin (rHb1.1) selectively inhibits vasorelaxation elicited by nitric oxide donors in rabbit isolated aortic rings.

We investigated the effect of a genetically engineered recombinant human hemoglobin (rHb1.1), specially designed to be used as a blood substitute, on the ability of various well-known vasodilators to relax the rabbit isolated aortic rings precontracted with the alpha-adrenoceptor agonist phenylephrine (PE) or with KCl (for nifedipine only). The vasorelaxant effects of nitroglycerin (NTG) and of sodium nitroprusside (SNP), two nitrovasodilators whose effects are mediated by nitric oxide (NO), were inhibited in a concentration-dependent manner by rHb1.1 (1.5 and 15 microM). Those elicited by isoproterenol, papaverine, histamine, adenosine, atriopeptin II, hydralazine, nifedipine, and cromakalim were comparatively little affected or not affected by rHb1.1 (15 microM). The ability of captopril to inhibit the vasoconstrictor action of angiotensin I (AT1) in the rabbit aortic rings was not reduced by rHb1.1 (15 microM). Our results suggest that rHb1.1 shares with purified human Hb the ability to inhibit selectively the vasorelaxant effect of NO-releasing substances such as NTG and SNP. Because the targeted plasma concentration of rHb1.1, when used as a blood substitute, is greater (approximately 50 times) than the highest concentration of rHb1.1 used in this study, significant drug interactions can be predicted between NO donors and rHb1.1.

Quantification of des-Arg9-bradykinin using a chemiluminescence enzyme immunoassay: application to its kinetic profile during plasma activation.

There is a renewed interest in the kininase I pathway of kinin metabolism, because des-Arg9-bradykinin (des-Arg9-BK) and des-Arg10-Lys-BK are selective and potent agonists of the B1 receptors, that are apparently upregulated by tissue injury. We have developed a polyclonal rabbit antiserum against des-Arg10-Lys-BK. In a radioimmunoassay for des-Arg10-Lys-BK, this antiserum exhibited high specificity. Notably, native kinins with the C-terminal Arg residue, bradykinin (BK) and Lys-BK, did not cross-react to a significant extent, whereas des-Arg9-BK and digoxigenin (DIG)-des-Arg9-BK exhibited a complete cross-reactivity. The antibodies were used to set up a sensitive chemiluminescence enzyme immunoassay (CLEIA) using the DIG-anti-DIG system as intermediate for the revelation of the immune complexes. The detection limit and the half-maximal saturation concentration for des-Arg9-BK were 27 and 1530 fmol/ml respectively. This assay, as well as another for BK quantification, have been applied in vitro to rabbit plasma activated by kaolin. The conversion of BK into des-Arg9-BK was generally efficient, and the persistence and concentration of both peptides were increased in the presence of enalaprilat an inhibitor of the angiotensin converting enzyme (ACEI). Rabbits treated with bacterial lipopolysaccharide exhibited an increase of plasma immunoreactive des-Arg9-BK that was potentiated in animals also treated with ACEI. This CLEIA for des-Arg9-BK is a new analytical tool applicable to analyze of the kininase I metabolites of kinins in vitro and in vivo. Measurements of des-Arg9-BK may be useful indicators of the kallikrein-kinin system activation.

Development of a binding assay for the B1 receptors for kinins.

A novel binding assay to kinin B1 receptors was developed, based on the design of a high-affinity agonist ligand, [125I]Tyr-Gly-Lys-Aca-Lys-des-Arg9-BK. Binding to rabbit aortic smooth muscle cells is highly temperature-dependent (optimal at 37 degrees C); apparent binding equilibrium is reached within 30 min, and competition by kinin analogs reveals the expected correlation with the B1 receptor pharmacology. The dissociation constant (Kd) of the labeled ligand is approx. 0.2 nM and this value does not change significantly as a function of cytokine pretreatment. However, the receptor abundance (Bmax) is significantly increased (1.5-fold) by pretreating the cells with interleukin-1 (IL-1), while oncostatin M (OSM) produces a marginal increase of the Bmax. This assay may be useful in documenting the regulation of B1 receptors in pathology.

Further analysis of the upregulation of bradykinin B1 receptors in isolated rabbit aorta by using metabolic inhibitors.

Kinins exert a contractile effect that develops as a function of the in vitro incubation time with isolated rabbit aorta. This response is mediated via receptors of the bradykinin B1 type and interleukin-1 amplifies this upregulation process. Tissues continuously treated with the protein synthesis inhibitor cycloheximide (71 microM) or with the protein trafficking inhibitor, brefeldin A (18 microM), failed to develop a contractile response to the bradykinin B1 receptor agonist, des-Arg9-bradykinin (1.7 microM) (72-100% inhibition of kinin response recorded at 3 or 6 h), whether or not they were exposed to interleukin-1 beta (290 pM). The protein glycosylation inhibitor tunicamycin exerted a selective and significant, but partial (50-76%), inhibition of des-Arg9-bradykinin-induced responses. The biochemical effect of the metabolic inhibitors on the tissue has been validated in assays involving incorporation of [3H]leucine and of [3H]mannose into protein or glycoprotein fractions, respectively. The modulatory effects of metabolic inhibitors on the responses to kinins of the isolated rabbit aorta support the idea that a de novo formation of membrane bradykinin B1 receptors is the molecular basis of both the spontaneous and the interleukin-1-stimulated upregulation phenomenon.

Recombinant human hemoglobin inhibits both constitutive and cytokine-induced nitric oxide-mediated relaxation of rabbit isolated aortic rings.

A genetically engineered recombinant human hemoglobin (rHb1.1) was recently developed for use as a blood substitute (Nature 1992;356:258-60). Like other mammalian hemoglobin (Hb) molecules, it might bind and antagonize the actions of nitric oxide (NO). We used an isolated rabbit aortic ring preparation to examine the ability of rHb1.1 to inhibit acetylcholine (ACh)- and interleukin-1 beta (IL-1 beta)-induced reductions of vasoconstrictor responses to the alpha-adrenoceptor agonist phenylephrine (PE). rHb1.1 (0.04-4.4 microM) rapidly and reversibly inhibited, in a concentration-dependent manner, both ACh- and IL-1 beta-induced decreases in PE contractile responses. These inhibitory effects of rHb1.1 were non-competitive and were equipotent to those of purified, cell-free human Hb (p.hHb). These two forms of soluble Hb were at least 10 times more potent than Hb in erythrocytes (red blood cells: RBC-Hb). Both NG-nitro-L-arginine (10 microM) a NO synthase inhibitor, and LY-83583 (10 microM), a guanylyl cyclase inhibitor, mimicked the effects of rHb1.1. The inhibitory effects of rHb1.1 were not shared by either human serum albumin (HSA 44 microM), which combines with but does not deactivate NO, or cytochrome C (44 microM), a heme-containing protein that does not bind NO; neither were they reversed by L-arginine (L-ARG) (1 mM), the presumed NO precursor. These and other results suggest that the chemical antagonism of NO is likely to be the mechanism by which rHb1.1 and other Hbs inhibit ACh- and IL-1 beta-induced decreases in the response to PE in rabbit aortic rings.