Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.     

Bradykinin receptors in isolated rat duodenum

Pharmacological properties of the bradykinin receptors in the isolated rat duodenum were investigated by examining the relaxant and contractile responses to bradykinin and [des-Arg9]-bradykinin, an agonist of B1 receptors. A specific desensitization and de novo formation for B1 receptors were observed. Changes in medium pH caused a decrease in the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum. Urea incubation in test tube inhibited the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum, while urea in bathing medium was ineffective. These findings strongly suggested that (a) ionic bonds are important in the interaction between bradykinin and its receptors, and (b) B2 receptors in rat duodenum are different from those in guinea pig ileum.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

The role of kinin B1 in the plasma extravasation of carrageenin-induced pleurisy

The role of des-Arg9-bradykinin (des-Arg9-BK) and kinin B1 receptor in the plasma extravasation of rat carrageenin-induced pleurisy was investigated employing B1 receptor agonist and antagonists and kininogen-deficient rats. Expression of the B1 receptor mRNA in pleura was induced from 3 to 5 h after the injection of carrageenin into the pleural cavity of Sprague-Dawley rats. Exogenous injection of des-Arg9-BK into the pleural cavity provoked a significant increase in plasma extravasation in 5 h carrageenin-induced pleurisy, but not in 20 min kaolin-induced pleurisy. The level of immunoreactive des-Arg9-BK in the exudate of 5 h carrageenin-induced pleurisy was higher than that of bradykinin (BK). Administration of the B1 receptor antagonists, des-Arg9-[Leu8]-BK or des-Arg9-D-Arg-[Hyp3, Thi5, D-Tic7,Oic8]-BK significantly reduced the exudation rate. However, intrapleural administration of des-Arg9-BK to plasma kininogen-deficient. Brown Norway-Katholiek rats did not result in a further increase in the plasma extravasation. In conclusion, endogenously generated des-Arg9-BK could contribute to the plasma extravasation in carrageenin-induced pleurisy via mediation of the inducible B1 receptor.     

Monoclonal antibodies to bradykinin inhibit smooth muscle contractile action of bradykinin

Four hybridoma cell lines have been established that secrete monoclonal antibodies to nonapeptide bradykinin. Bradykinin coupled to ovalbumin, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent, was used to immunize BALB/c mice. Spleen cells from the immunized animals were fused to P3-X63-AG8-653 mouse myeloma cells. The resultant hybrid cells were screened by enzyme-linked immunoassay for production of antibodies to bradykinin. Hybrids from four positive wells were subcloned by limiting dilution and expanded as ascites tumor into pristane-primed mice. All the four hybrids secreted monoclonal antibodies of IgG1 (k) isotype. Unlabeled peptides bradykinin, lysyl-bradykinin (kallidin) and methionyl-lysyl-bradykinin competed with the radiolabeled [Tyr1]kallidin for monoclonal antibody binding sites. These antibodies recognized preferentially either NH2- or COOH-terminals of the nonapeptide bradykinin and can distinguish between des-Arg1-bradykinin and des-Arg9-bradykinin. Bradykinin fragments smaller than eight residues were not recognized by these antibodies. Monoclonal antibodies BK-D6A5, BK-B6C9 and BK-A3D9 neutralized the smooth muscle contractile activity of bradykinin. An enzyme-linked immunoassay developed using these monoclonal antibodies showed the effective range of bradykinin determination between 5 and 150 ng.     

Bradykinin receptors: functional similarities in guinea pig gut muscle and mucosa

It is well established that bradykinin can stimulate mucosal electrolyte transport. However, the receptor type which mediates this effect has not been fully characterized. Recent studies have suggested that bradykinin and related kinins may act at two types of receptors designated as B1 and B2. We have determined the effect of bradykinin on electrolyte secretion across guinea pig ileal mucosa and longitudinal muscle in vitro in the presence and absence of D-Phe7-bradykinin (B2 antagonist) and des-Arg9-(Leu8)-bradykinin (B1 antagonist). The B2 antagonist (less than 100 microM) did not affect resting muscle tension or basal electrolyte transport but at 6-30 microM it caused a parallel rightward shift in the concentration-response curves to bradykinin in the mucosa (Ki = 4 microM) and muscle (Ki = 6 microM). Changes in electrolyte transport and muscle contractility evoked by bethanechol and substance P were not affected by the B2 antagonist (30 microM) in either the muscle or the mucosa. Moreover, changes in electrolyte transport and muscle contractility produced by bradykinin were not altered by the B1 antagonist (30 microM). Finally, the B1 agonist des-Arg9-bradykinin (10 nM-1 microM) was not active in either preparation. These data suggest that under normal conditions, ileal secretion and smooth muscle contractility in the guinea pig are regulated by B2-type bradykinin receptors.     

Neurotransmitter release from bradykinin-stimulated PC12 cells. Stimulation of cytosolic calcium and neurotransmitter release.

The effect of bradykinin on intracellular free Ca2+ and neurotransmitter secretion was investigated in the rat pheochromocytoma cell line PC12. Bradykinin was shown to induce a rapid, but transient, increase in intracellular free Ca2+ which could be separated into an intracellular Ca2+ release component and an extracellular Ca2+ influx component. The bradykinin-induced stimulation of intracellular free Ca2+ displayed a similar time course, concentration dependencies and extracellular Ca2+ dependence as that found for neurotransmitter release, indicating an association between intracellular free Ca2+ levels and neurotransmitter secretion. The selective BK1-receptor antagonist des-Arg9,[Leu8]BK (where BK is bradykinin) did not significantly affect the stimulation of intracellular free Ca2+ or neurotransmitter release. In contrast, these effects of bradykinin were effectively blocked by the selective BK2-receptor antagonist [Thi5,8,D-Phe7]BK, and mimicked by the BK2 partial agonist [D-Phe7]BK in a concentration-dependent manner. The stimulation of intracellular free Ca2+ and neurotransmitter release induced by bradykinin was shown not to involve voltage-sensitive Ca2+ channels, since calcium antagonists had no effect on either response at concentrations which effectively inhibit depolarization-induced responses. These results indicate that bradykinin, acting through the interaction with the BK2 receptor, stimulates an increase in intracellular free Ca2+ leading to neurotransmitter secretion. Furthermore, bradykinin-induced responses involve the release of intracellular Ca2+ and the influx of extracellular Ca2+ that is not associated with the activation of voltage-sensitive Ca2+ channels.     

Carboxypeptidase M and kinin B1 receptors interact to facilitate efficient b1 signaling from B2 agonists

Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg(9)-bradykinin or des-Arg(10)-kallidin. Carboxypeptidase M (CPM) is a membrane protein potentially well suited for this function. Here we show that CPM expression is required to generate a B1R-dependent increase in [Ca(2+)](i) in cells stimulated with B2R agonists kallidin or bradykinin. CPM and the B1R interact on the cell membrane, as shown by co-immunoprecipitation, cross-linking, and fluorescence resonance energy transfer analysis. CPM and B1R are also co-localized in lipid raft/caveolin-enriched membrane fractions, as determined by gradient centrifugation. Treatment of cells co-expressing CPM and B1R with methyl-beta-cyclodextrin to disrupt lipid rafts reduced the B1R-dependent increase in [Ca(2+)](i) in response to B2R agonists, whereas cholesterol treatment enhanced the response. A monoclonal antibody to the C-terminal beta-sheet domain of CPM reduced the B1R response to B2R agonists without inhibiting CPM. Cells expressing a novel fusion protein containing CPM at the N terminus of the B1R also increased [Ca(2+)](i) when stimulated with B2R agonists, but the response was not reduced by methyl-beta-cyclodextrin or CPM antibody. A B1R- and CPM-dependent calcium signal in response to B2R agonist bradykinin was also found in endothelial cells that express both proteins. Thus, a close relationship of B1Rs and CPM on the membrane is required for efficiently generating B1R signals, which play important roles in inflammation.     

Identification of a new kinin in human urine

The types of kinins excreted in fresh urine of dogs, rats, and humans were compared. Urinary kinins were separated by reverse-phase (C18) high performance liquid chromatography and quantitated by radioimmunoassay using an antibody directed against the COOH-terminal region of the peptide. Kinins were found in the following proportions: 53 +/- 3% bradykinin, 23 +/- 4% Lys-bradykinin, and 13 +/- 7% des-Arg1-bradykinin in dog urine; 67 +/- 6% bradykinin, 6 +/- 3% Lys-bradykinin, and 10 +/- 3% des-Arg1-bradykinin in rat urine; and 12 +/- 4% bradykinin, 30 +/- 3% Lys-bradykinin, 2 +/- 1% des-Arg1-bradykinin, and 41 +/- 3% unknown kinin in human urine. The unknown kinin was purified from a pool of human urine. Amino acid sequencing revealed a structure similar to Lys-bradykinin except that proline in position 4 was replaced by alanine ([Ala3]Lys-bradykinin). Synthetic and endogenous [Ala3]Lys-bradykinins had similar high performance liquid chromotography elution volumes and both had vasodilator activity and contracted the rat uterus. Human urinary kallikrein incubated with semipurified human low molecular weight kininogen released 76% of the total kinins as Lys-bradykinin, 7% as bradykinin, and 17% as [Ala3]Lys-bradykinin. In contrast, rat urinary kallikrein released 86% bradykinin, 18% Lys-bradykinin, and negligible amounts of [Ala3]Lys-bradykinin. The study revealed the presence of a new kinin, [Ala3]Lys-bradykinin, in human urine and it also proves that the types of kinins generated intrarenally are species-dependent.     

Bradykinin receptors in intestinal smooth muscles and their post-receptor events related to calcium

The effects of trifluoperazine and verapamil on bradykinin- and des-Arg(9)-bradykinin induced responses of isolated rat duodenum and guinea-pig ileum were investigated to elucidate post-bradykinin receptor events. Verapamil and trifluoperazine inhibited bradykinin induced relaxations and contractions and des-Arg(9)- bradykinin induced contractions in rat duodenum. Bradykinin induced contractions of ileum were also inhibited by trifluoperazine and. verapamil. Since non-competitive affinity constants of trifluoperazine and verapamil for the relaxant responses to bradykinin in duodenum and for the contractile responses to bradykinin in ileum are different, post-bradykinin receptor events related to calcium may be different in ileum and duodenum. In addition, affinity constants of bradykinin in guinea-pig ileum and rat duodenum are also disparate suggesting the presence of different types of bradykinin B(2) receptors in these two organs.     

5-hydroxytryptamine-evoked [Ca]i oscillations in rat C6 glioma cells

We have investigated the modulation of the intracellular calcium concentration ([Ca²⁺]_i) in rat C6 glioma cells following their activation by the agonists 5-hydroxytryptamine·HCl (5-HT) and bradykinin, using single cell imaging of [Ca²⁺]_i with the calcium-sensitive dye Fura-2. The majority of the signals observed involved release of calcium from intracellular stores, and after prolonged application of 5-HT, but not bradykinin, the cells exhibited oscillations in [Ca²⁺]_i levels. These calcium oscillations were dependent on the presence of extracellular calcium, and were unaffected by the calcium channel antagonists nifedipine and verapamil. Caffeine, which in other cell types is able to release calcium from inositol trisphosphate-insentive stores, had very little effect on [Ca²⁺]_i levels in C6 cells. On the other hand, bradykinin, although able to elevate [Ca²⁺]_i probably by acting via the B₂-receptor subtype, was unable to induce any calcium oscillations in these cells.     

Bradykinin modulates the ouabain-insensitive Na+-ATPase activity from basolateral membrane of the proximal tubule.

This paper studies the modulation by bradykinin of the ouabain-insensitive Na+-ATPase activity in both renal cortex homogenate and basolateral membrane from proximal tubule. The increase in bradykinin concentration from 10-14 to 10-10 M stimulated the ouabain-insensitive Na+-ATPase activity in cortex homogenates about 2.2-fold, but inhibited the enzyme activity of basolateral membrane preparations by 60%. In both preparations, the maximal effect was obtained with 10-10 M bradykinin. Further increase in the concentration of bradykinin completely abolished these effects. The antagonist of the B2 receptor, Hyp3, completely abolished the effect of 10-10 M bradykinin on the Na+-ATPase activity in the basolateral membrane preparation in a dose-dependent manner, but had no effect on the bradykinin stimulated enzyme activity of the cortex homogenate. Furthermore, in the presence of 10-7 M Hyp3, 10-10 M bradykinin stimulated the Na+-ATPase activity by 45% in the basolateral membrane preparations. The increase in des-Arg9-bradykinin concentration from 10-12 to 10-7 M, an agonist of the B1 receptor, stimulated the Na+-ATPase activity of the cortex homogenates and of the basolateral membrane preparations by 105 and 148%, respectively. In the presence of 25 microM mergetpa, an inhibitor of kininase I, the increase in bradykinin concentration from 10-12 to 10-10 M promoted similar inhibition of the Na+-ATPase activity of both cortex homogenates and basolateral membrane preparations. These results suggest that bradykinin stimulated the Na+-ATPase activity of proximal tubule through the interaction with B1 receptors and inhibited the enzyme through the interaction with B2 receptors. Furthermore, the cortex homogenate expresses a kininase I activity that cleaves bradykinin to des-Arg9-bradykinin.     

Kinin B1 receptor-mediated motor responses in normal or inflamed rat urinary bladder in vivo

The rat urinary bladder is one of the few in vivo preparations in which kinin B1 receptor-mediated contractile responses have been described, but the nature (local or reflex) of these responses has not been characterized. We have investigated the motor effects of i.v. or topical (onto the bladder serosa) administration of the selective kinin B1 receptor agonist [des-Arg9]-bradykinin ([des-Arg9]-BK) in the normal or inflamed (cyclophosphamide-induced) urinary bladder in urethane-anaesthetized rats. In both normal and inflamed bladders [des-Arg9]-BK produced a tonic contraction of low amplitude (< 15 mmHg) with phasic contractions of high amplitude (> or = 15 mmHg) superimposed (micturition reflex contractions). In inflamed bladders, the response to [des-Arg9]-BK was more prominent than in controls. Similar observations were made after the topical administration of [des-Arg9]-BK. In order to evaluate any time-dependency in the expression of B1 receptor-mediated bladder responses, [des-Arg9]-BK was administered in separate groups of control animals at 30 and 240 min after the completion of surgical procedures required for set-up of the preparation: no bladder contraction was detected at 30 min whereas both local and reflex contractions could be elicited by [des-Arg9]-BK at 240 min after the set up. In ganglionectomized rats, the response to [des-Arg9]-BK or the selective tachykinin NK2 receptor agonist [betaAla8]NKA(4-10) was evaluated at 30 and 240 min after the set up in inflamed or in control animals. The response to [des-Arg9]-BK was greater after inflammation although a time-dependent increase was evident in both groups; in contrast, the response to [betaAla8]NKA(4-10) was similar in both groups and remained constant over the observation period. After induction of inflammation, the tonic contraction induced by [des-Arg9]-BK in ganglionectomized rats was dose-dependently reduced by the kinin B1 receptor antagonist [desArg10]Hoe 140. The contractile response (number of micturition reflex contractions) induced by [des-Arg9]-BK in normal rats with intact pelvic nerves at 240 min from the set up was not changed after the administration of the selective B2 receptor antagonist Hoe 140. These results indicate that stimulation of bladder kinin B1 receptors evokes a local, tonic-type contraction with reflex contractions superimposed in both normal and inflamed bladders, but in the latter situation the motor responses are magnified.     

Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: a possible role for tyrosine kinases

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/L (Des-Arg(9))-Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation.     

Characterization of Bradykinin-Induced Phosphoinositide Turnover in Neurohybrid NCB-20 Cells

Phosphoinositide hydrolysis was studied in neurohybrid NCB-20 cells prelabeled with myo-[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IP1) in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 microM. The bradykinin response was robust (10-fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D-Arg-(Hyp3, Thi, D-Phe)-bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine H1 receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin-induced [3H]IP1 accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carbachol-induced accumulation in the same cell line. In contrast, using related neurohybrid NG108-15 cells, bradykinin-induced [3H]IP1 accumulation was found to require much less lithium. IN the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]-inositol bis- and trisphosphate. Basal and bradykinin-induced phosphoinositide breakdown was inhibited by 4 beta-phorbol 12,13-dibutyrate, but was unaffected by the biologically inactive 4 beta-phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the bradykinin-induced [3H]IP1 accumulation, without affecting basal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The renin-angiotensin and the kallikrein-kinin systems

The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) each encompasses a large number of molecules, with several participating in both systems. The RAS generates a family of bioactive angiotensin peptides with varying biological activities. These include angiotensin-(1-8) (Ang II), angiotensin-(2-8) (Ang III), angiotensin-(3-8) (Ang IV), and angiotensin-(1-7) [Ang-(1-7)]. Ang II and Ang III act on type 1 (AT(1)) and type 2 (AT(2)) angiotensin receptors, whereas, Ang IV and Ang-(1-7) act on their own receptors. The KKS also generates a family of bioactive peptides with varying biological activities. These include hydroxylated and non-hydroxylated bradykinin and kallidin peptides and their carboxypeptidase metabolites des-Arg(9)-bradykinin and des-Arg(10)-kallidin. Whereas bradykinin and kallidin act mainly via the type 2 bradykinin (B(2)) receptor, des-Arg(9)-bradykinin and des-Arg(10)-kallidin act mainly via the type 1 bradykinin (B(1)) receptor. The AT(1) receptor forms heterodimers with the AT(2) and B(2) receptors and there is cross talk between the AT(1) and epidermal growth factor receptors. The B(2) receptor also interacts with angiotensin converting enzyme and nitric oxide synthase. Both angiotensin and kinin peptides are metabolised by many different peptidases that are important determinants of the activities of the RAS and KKS, and several of which participate in both systems.     

B(1) and B(2) bradykinin receptors on adventitial fibroblasts of cerebral arteries are coupled to recombinant eNOS

Our previous ex vivo and in vivo studies reported that expression of the recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts recovers NO production in arteries without endothelium in response to bradykinin. The present study was designed to characterize subtypes of bradykinin receptors on adventitial fibroblasts coupled to the activation of recombinant eNOS. Endothelium-denuded segments of canine basilar arteries were transduced with beta-galactosidase (beta-Gal) gene or eNOS gene ex vivo, using a replication-defective adenoviral vector (10(10) plaque-forming units/ml) for 30 min at 37 degrees C. Twenty-four hours later, isometric force recording or cGMP measurement was carried out. B(1) bradykinin receptor agonist (des-Arg(9)-bradykinin, 10(-10)-10(-8) mol/l) did not significantly affect vascular tone in control or beta-Gal gene-transduced canine basilar arteries without endothelium. In contrast, this agonist caused concentration-dependent relaxations in recombinant eNOS gene-transduced arteries without endothelium. Relaxations to B(1) receptor agonist in the eNOS arteries were abolished by B(1) receptor antagonist (des-Arg(9)-[Leu(8)]bradykinin, 6 x 10(-9) mol/l) but not by B(2) receptor antagonist (Hoe-140, 5 x 10(-8) mol/l). Bradykinin did not significantly alter vascular tone in control or beta-gal arteries without endothelium, whereas this peptide (10(-11)-10(-8) mol/l) induced concentration-dependent relaxations, as well as an increase in cGMP formation in endothelium-denuded eNOS-transduced arteries. Stimulatory effects of bradykinin were prevented in the presence of a B(2) receptor antagonist but not in the presence of a B(1) receptor antagonist. B(1) and B(2) receptor antagonists had no effect on relaxations to substance P, confirming the selectivity of the compounds. Our results suggest that B(1) and B(2) bradykinin receptors are coupled to activation of recombinant eNOS expressed in adventitial fibroblasts.     

Structure-activity studies of [des-Arg9]-bradykinin on the B1 receptor of the rabbit aorta.

Eight L-alanine analogues of [des-Arg9]-bradykinin and a few other compounds substituted in positions 5 and (or) 8 have been tested on rabbit aortic strips in order to identify the group(s) responsible for binding and (or) stimulation of the B1 receptor. The results obtained with the L-Ala series have shown that the active group is located at the C-terminal end and it is probably Phe8, while the middle part and the N-terminal end of the peptide molecule are primarily involved in binding the agonist to the receptor. An aromatic ring is required in position 8 for activation of receptors, since the elimination or aromaticity (as in [Leu8,des-Arg9]-bradykinin and in [cyclohexylalanine8,des-Arg9]-bradykinin) brings about pure and competitive antagonists. Some compounds exert an angiotensin-like effect when applied at very high concentrations.     

Conformation-activity relations of des-Arg9-bradykinin. I. Conformation of molecules in solution

The spatial structure of [des-Arg9]bradykinin, a selective agonist of the B1 type kinin receptors, was studied by means of theoretical conformational analysis. In order to restrict the number of conformations under consideration, we used available data on 1H NMR in (CD3)2SO solution indicating the C-terminal carboxyl group to interact with the guanidine group of Arg1 and the hydroxyl group of Ser6. The calculated set of [des-Arg9]bradykinin low-energy conformations was compared with the 1H NMR data. Four types of conformations were selected, which are consistent with experimental data and serve as sterically reliable models for the preferable three-dimensional structure of [des-Arg9]bradykinin in solution.     

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED50 for this stimulation is 4.52 +/- 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [3H]thymidine incorporation in the NIL8 cells, whereas desarg9-bradykinin is without effect, suggesting action of the kinin peptides through type B2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE1, PGE2, PGA1, PGA2, PGB1, and PGB2 strongly inhibit [3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.