Cross talk between beta-adrenergic and bradykinin B(2) receptors results in cooperative regulation of cyclic AMP accumulation and mitogen-activated protein kinase activity.

Costimulation of G protein-coupled receptors (GPCRs) may result in cross talk interactions between their downstream signaling pathways. Stimulation of GPCRs may also lead to cross talk regulation of receptor tyrosine kinase signaling and thereby to activation of mitogen-activated protein kinase (MAPK). In COS-7 cells, we investigated the interactions between two particular mitogenic receptor pathways, the endogenously expressed beta-adrenergic receptor (beta-AR) and the transiently transfected human bradykinin (BK) B(2) receptor (B(2)R). When beta-AR and B(2)R are costimulated, we found two different cross talk mechanisms. First, the predominantly G(q) protein-coupled B(2)R is enabled to activate a G(i) protein and, subsequently, type II adenylate cyclase. This results in augmentation of beta-AR-mediated cyclic AMP (cAMP) accumulation by BK, which alone is unable to increase the cAMP level. Second, independently of BK-induced superactivation of the cAMP system, costimulation of beta-AR leads to protein kinase A-mediated blockade of phospholipase C activation by BK. Thereby, the pathway from B(2)R to MAPK, which essentially involves protein kinase C activation, is selectively switched off. The MAPK activation in response to isoproterenol was not affected due to costimulation. Furthermore, in the presence of isoproterenol, BK lost its ability to stimulate DNA synthesis in COS-7 cells. Thus, our findings might establish a novel paradigm: cooperation between simultaneously activated mitogenic pathways may prevent multiple stimulation of MAPK activity and increased cell growth.

Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity.

G protein-coupled receptors (GPCRs) can stimulate the mitogen-activated protein kinase (MAPK) cascade and thereby induce cellular proliferation like receptor tyrosine kinases (RTKs). Work over the past 5 years has established several models which reduce the links of G(i)-, G(q)-, and G(s)-coupled receptors to MAPK on few principle pathways. They include (i) Ras-dependent activation of MAPK via transactivation of RTKs such as the epidermal growth factor receptor (EGFR), (ii) Ras-independent MAPK activation via protein kinase C (PKC) that converges with the RTK signalling at the level of Raf, and (iii) activation as well as inactivation of MAPK via the cAMP/protein kinase A (PKA) pathway in dependency on the type of Raf. Most of these generalizing hypotheses are founded on experimental data obtained from expression studies and using a limited set of individual receptors. This review will compare these models with pathways to MAPK found for a great variety of peptide hormone and neuropeptide receptor subtypes in various cells. It becomes evident that under endogenous conditions, the transactivation pathway is less dominant as postulated, whereas pathways involving isoforms of PKC and, especially, phosphoinositide 3-kinase (PI-3K) appear to play a more important role as assumed so far. Highly cell-specific and unusual connections of signalling proteins towards MAPK, in particular tumour cells, might provide points of attacks for new therapeutic concepts.

Bradykinin signalling to MAP kinase: cell-specific connections versus principle mitogenic pathways.

Mitogenic signalling pathways from G protein-coupled receptors (GPCRs) to the mitogen-activated protein kinase (MAPK) cascade may involve alpha- or betagamma-subunits of heterotrimeric G proteins, receptor or non-receptor tyrosine kinases, adaptor molecules, phosphoinositide 3-kinases, protein kinase C, and probably other proteins. The majority of models describing the connection of different signalling proteins within a mitogenic pathway are based on experimental data obtained by co- and overexpression of epitope-tagged MAPK together with the respective GPCR and other signalling proteins of interest in transfectable cell lines. Here the link of the bradykinin B2 receptor (B2R) to MAPK in the COS-7 cell expression system is compared with mitogenic signalling pathways of bradykinin in various tumour cell lines. It becomes evident that in natural or tumour cells expressing individual amounts and different isoforms of signalling proteins completely other relations between B2R and MAPK may exist than in COS-7 cells, suggesting a high degree of cellular specificity in mitogenic signalling.

Signal transduction pathways of G protein-coupled receptors and their cross-talk with receptor tyrosine kinases: lessons from bradykinin signaling.

G protein-coupled receptors (GPCRs) represent a major class of drug targets. Recent investigation of GPCR signaling has revealed interesting novel features of their signal transduction pathways which may be of great relevance to drug application and the development of novel drugs. Firstly, a single class of GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of G proteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutive initiation of different signaling chains. Secondly, the different signaling pathways emanating from one or several GPCRs exhibit extensive cross-talk, resulting in positive or negative signal modulation. Thirdly, GPCRs including B2R have the capacity for generation of mitogenic signals. GPCR-induced mitogenic signaling involves activation of the p44/p42 "mitogen activated protein kinases" (MAPK) and frequently "transactivation" of receptor tyrosine kinases (RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partly understood pathways. Cytoplasmic tyrosine kinases and protein-tyrosine phosphatases (PTPs) which regulate RTK signaling are likely mediators of RTK transactivation in response to GPCRs. Finally, GPCR signaling is the subject of regulation by RTKs and other tyrosine kinases, including tyrosine phosphorylation of GPCRs itself, of G proteins, and of downstream molecules such as members of the protein kinase C family. In conclusion, known agonists of GPCRs are likely to have unexpected effects on RTK pathways and activators of signal-mediating enzymes previously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs may be of much interest for modulating GPCR-mediated biological responses.

Expression and functional characterization of a pHis-tagged human bradykinin B2 receptor in COS-7 cells.

A polyHis-tagged bradykinin (BK) B2 receptor (pHis-BKR) cDNA was constructed and expressed in COS-7 cells. The pHis-BKR is suitable for both immunoprecipitation and immunoblotting with anti-polyHis antibodies and can be easily purified using Ni-NTA columns. Immunochemical detection revealed a molecular mass of approximately 66 kDa. The pHis-BKR is capable of mediating BK-induced stimulation of inositol phosphate formation as well as of mitogen-activated protein kinase (MAPK) activity. Compared with the wild-type receptor (WT-BKR) the tagged receptor showed a slightly enhanced affinity towards BK but a reduced expression level. Despite these modified pharmacological properties the pHis-tagged BKR may be a useful tool for studying BKR modifications and signaling.

Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells.

Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR.

In various tumour cell lines the peptide bradykinin B(2) receptor antagonist, Hoe 140 (Icatibant), may act as mitogenic agonist.

This study examined the mitogenic effects of bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), the peptide bradykinin B(2) receptor antagonist Hoe 140 (D-Arg(0)[Hyp(3)-Thi(6)-D-Tic(7)-Oic(8)]BK, and the orally active, nonpeptide B(2) receptor antagonist FR 173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-2-4-dichloro-3-[(2-methyl-8-quino linyl) oxymethyl]phenyl]-N-methylaminocarbonyl-methyl]acrylamide) in three different human tumour cell lines: the small cell lung carcinoma (SCLC) cell line H-69, the breast carcinoma cell line EFM-192A, and the colon carcinoma cell line SW-480. In these cell lines activation of mitogen-activated protein kinase (MAPK) is involved in BK-induced stimulation of cell proliferation and may be mediated by both G(q) proteins (SW-480) and G(i) proteins (EFM-192A; H-69). In these cells BK as well as Hoe 140 increased the rate of DNA synthesis measured with the [(3)H]-thymidine uptake assay. Hoe 140 did neither antagonize nor potentiate the effect of BK. FR 173657 did not stimulate [(3)H]-thymidine incorporation but clearly antagonized the mitogenic effects of BK as well as Hoe 140. In H-69 cells, FR 173657 induced a decrease in the basal rate of DNA synthesis. In all three cell lines BK and Hoe 140 stimulated the activity of MAPK. Their effect on MAPK activity was completely abolished by FR 173657 which itself did not increase the activity of MAPK. In H-69 cells, the basal activity of MAPK was slightly inhibited by FR 173657. In the cell lines SW-480 and H-69 both BK and Hoe 140 but not FR 173657 stimulated phosphatidylinositol hydrolysis. In H-69 cells, FR 173657 decreased basal inositol phosphate formation. Our results show that in certain tumour cell lines the classical peptide B(2) receptor antagonist, Hoe 140, may act as mitogenic B(2) receptor agonist whereas the nonpeptide B(2) receptor antagonist, FR 173657, does not. In H-69 cells FR 173657 was found to exhibit properties of an inverse agonist.

Bradykinin B(2) receptor-mediated mitogen-activated protein kinase activation in COS-7 cells requires dual signaling via both protein kinase C pathway and epidermal growth factor receptor transactivation.

The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.

A novel mitogenic signaling pathway of bradykinin in the human colon carcinoma cell line SW-480 involves sequential activation of a Gq/11 protein, phosphatidylinositol 3-kinase beta, and protein kinase Cepsilon.

The signaling routes connecting G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) pathway reveal a high degree of complexity and cell specificity. In the human colon carcinoma cell line SW-480, we detected a mitogenic effect of bradykinin (BK) that is mediated via a pertussis toxin-insensitive G protein of the Gq/11 family and that involves activation of MAPK. Both BK-induced stimulation of DNA synthesis and activation of MAPK in response to BK were abolished by two different inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY 294002, as well as by two different inhibitors of protein kinase C (PKC), bisindolylmaleimide and Ro 31-8220. Stimulation of SW-480 cells by BK led to increased formation of PI3K lipid products (phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 4-bisphosphate) and to enhanced translocation of the PKCepsilon isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110beta and p85, but not p110alpha and p110gamma, were detected in SW-480 cells. Finally, p110beta was found to be co-immunoprecipitated with PKCepsilon. Our data suggest that in SW-480 cells, (i) dimeric PI3Kbeta is activated via a Gq/11 protein; (ii) PKCepsilon is a downstream target of PI3Kbeta mediating the mitogenic signal to the MAPK pathway; and (iii) PKCepsilon associates with the p110 subunit of PI3Kbeta. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.

Bradykinin antagonists with dehydrophenylalanine analogues at position 5.

Continuing the studies on structural requirements of bradykinin antagonists, it has been found that analogues with dehydrophenylalanine (deltaPhe) or its ring-substituted analogues (deltaPhe(X)) at position 5 act as antagonists on guinea pig pulmonary artery, and on guinea pig ileum. Because both organs are considered to be bradykinin B2 receptor tissues, the analogues with deltaPhe or deltaPhe(X) at position 5, but without any replacement at position 7, seem to represent a new structural type of B2 receptor antagonist. All the analogues investigated act as partial antagonists; they inhibit the bradykinin-induced contraction at low concentrations and act as agonists at higher concentrations. Ring substitutions by methyl groups or iodine reduce both the agonistic and antagonistic activity. Only substitution by fluorine gives a high potency. Incorporation of deltaPhe into different representative antagonists with key modifications at position 7 does not enhance the antagonist activity of the basic structures, with one exception. Only the combination of deltaPhe at position 5 with DPhe at position 7 increases the antagonistic potency on guinea pig ileum by about one order of magnitude. Radioligand binding studies indicate the importance of position 5 for the discrimination of B2 receptor subtypes. The binding affinity to the low-affinity binding site (KL) was not significantly changed by replacement of Phe by deltaPhe. In contrast, ring-methylation of deltaPhe results in clearly reduced binding to KL. The affinity to the high-affinity binding site (KH) was almost unchanged by the replacement of Phe in position 5 by deltaPhe, whereas the analogue with 2-methyl-dehydrophenylalanine completely failed to detect the KH-site. The peptides were synthesized on the Wang-resin according to the Fmoc/Bu(t) strategy using Mtr protection for the side chain of Arg. The dehydrophenylalanine analogues were prepared by a strategy involving PyBop couplings of the dipeptide unit Fmoc-Gly-deltaPhe(X)-OH to resin-bound fragments.

Novel bradykinin signalling events in PC-12 cells: stimulation of the cAMP pathway leads to cAMP-mediated translocation of protein kinase Cepsilon.

In the rat pheochromocytoma cell line PC-12, bradykinin (BK) stimulated phosphatidylinositol hydrolysis by 4-5-fold and, additionally, intracellular cAMP accumulation by approx. 1.6-fold. EC50 values for BK were 3 nM and 2 nM respectively. The BK-induced increase in cAMP accumulation was paralleled by a 1.6-fold increase in protein kinase A (PKA) activity. The time course of BK-stimulated inositol phosphate formation was rapid (t1/2<1 min), whereas the BK-induced cAMP accumulation was lagging (t1/2 approx. 6 min). The effect of BK on the cAMP pathway was independent of pertussis toxin, excluding an indirect stimulation of adenylate cyclase via betagamma-complexes from Gi or Go proteins. Two different protein kinase C (PKC) inhibitors, bisindolylmaleimide and Ro 31-820, failed to prevent BK-induced cAMP accumulation, and exclude PKC as mediator of BK action on adenylate cyclase. In contrast, the stimulatory effect of BK on cAMP accumulation was completely abolished by two calmodulin antagonists, chlorpromazine and ophiobolin, suggesting an indirect, Ca2+/calmodulin-mediated effect of BK on the cAMP pathway. In addition, exposure of PC-12 cells to BK resulted in a translocation of the PKC isoforms alpha, delta, epsilon and zeta displaying different kinetics. The BK-induced translocations of the PCDs alpha and delta were rapid and biphasic, whereas the PKCs epsilon and zeta revealed a slower and slightly transient translocation in response to BK. The BK-elicited translocation of PKCepsilon, but not that of the PKCs alpha, delta and zeta, was prevented by two different inhibitors of adenylate cyclase, 2',5'-dideoxyadenosine and MDL-12,330A, as well as the PKA inhibitor adenosine 3':5'-monophosphothioate. These findings suggest that the BK-induced translocation of novel (n)PKCepsilon is mediated via the cAMP pathway. Since nPKCepsilon appears to regulate neurite outgrowth in PC-12 cells [Hundke, McMahon, Dadgar and Messing (1995) J. Biol. Chem. 270, 30134-30140] our results provide evidence for a novel signalling mechanism that might be involved in BK-induced neuronal differentiation of PC-12 cels.

Signaling effects of alpha-thrombin and SFLLRN in rat glioma C6 cells.

Effects of thrombin on brain cells, including change of neurite outgrowth and astrocyte shape, are described, but the molecular mechanisms are unclear. We investigated the effects of human alpha-thrombin and a six amino acid thrombin receptor activating peptide (TRAP-6, SFLLRN) on [Ca2+]i, phosphoinositide hydrolysis, and protein kinase C in rat glioma C6 cells. Stimulation of C6 cells with both alpha-thrombin and TRAP-6 resulted in [Ca2+]i mobilization, [3H]Inositol phosphate response, and enhanced immunoreactivity of the protein kinase C (PKC) isoenzymes alpha, beta, gamma, delta, and epsilon. Results suggest that alpha-thrombin and TRAP-6 activate at least partially the same intracellular signaling pathways in rat glioma C6 cells, which is evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma C6 cells.

Tyrosine phosphorylation of GSalpha and inhibition of bradykinin-induced activation of the cyclic AMP pathway in A431 cells by epidermal growth factor receptor.

An increasing amount of experimental data suggest that cross-talk exists between pathways involving tyrosine kinases and heterotrimeric G proteins. In a previous study, we demonstrated that bradykinin (BK) increases the intracellular accumulation of cAMP in the human epidermoid carcinoma cell line A431 by stimulating adenylate cyclase activity via a stimulatory G protein (Gsalpha) (Liebmann, C., Graness, A., Ludwig, B., Adomeit, A., Boehmer, A., Boehmer, F.-D., Nürnberg, B., and Wetzker, R. (1996) Biochem. J. 313, 109-118). Here, we present several lines of evidence indicating the ability of epidermal growth factor (EGF) to suppress BK-induced activation of the cAMP pathway in A431 cells via tyrosine phosphorylation of Gsalpha. Gsalpha was specifically immunoprecipitated from A431 cells using the anti-alphas antiserum AS 348. Tyrosine phosphorylation of Gsalpha was detectable in EGF-pretreated cells with monoclonal anti-phosphotyrosine antibodies. Additionally, A431 cells were labeled with [32P]orthophosphate in vivo and treated with EGF, and the resolved immunoprecipitates were subjected to amino acid analysis. The results clearly indicate that EGF induces tyrosine phosphorylation of Gsalpha in A431 cells. Treatment of A431 cells with EGF decreased BK-induced cAMP accumulation in intact cells as well as the stimulation of adenylate cyclase by BK, NaF, and guanyl nucleotides, but not by forskolin. Also, EGF treatment abolished both the BK- and isoprenaline-induced stimulation of guanosine 5'-O-(3-[35S]thiotriphosphate) binding to Gsalpha. In contrast, the BK-evoked, Gq-mediated stimulation of inositol phosphate formation in A431 cells was not affected by EGF pretreatment. Thus, EGF-induced tyrosine phosphorylation of Gsalpha is accompanied by a loss of its susceptibility to G protein-coupled receptors and its ability to stimulate adenylate cyclase via guanyl nucleotide exchange. We propose that Gsalpha may represent a key regulatory protein in the cross-talk between the signal transduction pathways of BK and EGF in A431 cells.

Highly selective bradykinin agonists and antagonists with replacement of proline residues by N-methyl-D- and L-phenylalanine.

For further studies on the structural and conformational requirements of positions 2,3, and 7 in the bradykinin sequence, we replaced the proline residues by the more hydrophobic and conformationally restricted N-methyl-L- and D-phenylalanine (NMF). The biological activities of the new analogs were evaluated on rat uterus, guinea pig ileum, and guinea pig lung strip. Receptor binding of the analogs was studied in membranes from rat uterus and guinea pig ileum. Influence of bradykinin analogs on the release of cytokines from mouse spleen cell cultures was also measured. Bradykinin analogs were synthesized by the solid phase method, using Boc strategy on PAM or Merrifield resins. The best results in the formation of the N-methylamide bond were obtained with the coupling reagent PyBrop. In position 7 the substitution of D-Phe by D-NMF, retaining the configuration of the amino acid, converts bradykinin antagonists into agonists. The bradykinin analogs with D-NMF at position 7 gave the highest known tissue selectivity for rat uterus among agonists. [L-NMF(2)]bradykinin has moderate agonist activity on rat uterus but antagonist activity on guinea pig lung strip. It represents a new antagonist for B(2) receptors without any replacement at position 7. The same analog completely inhibits bradykinin-evoked cytokine expression by mononuclear cells.

Dual bradykinin B2 receptor signalling in A431 human epidermoid carcinoma cells: activation of protein kinase C is counteracted by a GS-mediated stimulation of the cyclic AMP pathway.

Cell membranes of the human epidermoid cell line A431 express classical bradykinin (BK) B2 receptors, as assessed by [3H]BK binding studies. Furthermore, stimulation by BK induced a time-dependent modulation of protein kinase C (PKC) activity in A431 cells: a rapid activation (t1/2 approximately 1 min) is followed by a slow inhibition (t1/2 approximately 20 min) of PKC translocation measured by [3H]phorbol 12,13-dibutyrate binding. In addition, BK stimulated both adenylate cyclase activity in A431 membranes and accumulation of intracellular cyclic AMP (cAMP) in intact cells in a retarded manner. A possible BK-induced activation of the cAMP pathway mediated via PKC, phospholipase D, prostaglandins or Ca2+/calmodulin was excluded. A 35 kDa protein was found in A431 membranes to be specifically phosphorylated in the presence of both BK and protein kinase A (PKA). An anti-alpha s-antibody, AS 348, abolished stimulation of adenylate cyclase activity in response to BK, cholera toxin and isoprenaline, strongly suggesting the involvement of Gs proteins in the BK action. The BK-activated cAMP signalling system might be important for the observed inactivation of PKC slowly evoked by BK: the BK-induced rapid activation of PKC is decreased by dibutyryl cAMP, and the slow inhibition of PKC is prevented by an inhibitor of PKA, adenosine 3':5'-monophosphothioate (cyclic, Rp isomer). The inhibition of PKC translocation might be exerted directly at the level of PKC activation, since stimulation of phosphoinositide hydrolysis by BK was affected by neither dibutyryl cAMP nor forskolin. Thus our results provide the first evidence that A431 cells BK is able to activate two independent signal-transduction pathways via a single class of B2 receptors but two different G proteins. The lagging stimulation of the cAMP signalling pathway via Gs might serve to switch off PKC, which is rapidly activated via Gq-mediated stimulation of phosphoinositide hydrolysis.

New cyclic bradykinin antagonists containing disulfide and lactam bridges at the N-terminal sequence.

Continuing our studies of the bioactive conformation of bradykinin (BK) antagonists, we synthesized a first series of analogues with side-chain cyclization in the N-terminal sequence. Through this conformational constraint it should be possible to gain insight into their three-dimensional structure. The cycles were proposed on the basis of existing ideas and hypotheses about the receptor bound conformation of BK and its antagonists. The reported peptides contain D-Phe at position 7 or D-Tic-Oic (D-Tetrahydroisoquinoline-3 -carboxyl-octahydroindole-2-carboxylic acid) at positions 7 and 8, respectively, and a disulfide or lactam bridge between positions 0 and 6. Syntheses, including cyclization reactions, were carried out on PAM resin. The biological activity of the lead compound [DPhe7]-BK, the linear precursors and the cyclic peptides, as estimated on isolated rat uterus, guinea pig ileum and lung strips, are in the same range. The conformational properties of the new cyclic analogues were studied through energy minimization on a model compound. The results of the calculations support the existence of low-energy structures containing a beta-turn. Therefore, such a turn in the N-terminal segment of the molecule can be proposed as an important structural feature of the bioactive conformation of BK antagonists.

The adenylate cyclase-inhibiting bradykinin receptor in guinea pig ileum membranes exhibits an unique antagonist profile.

The purpose of the present study was to characterize more precisely an inhibitory, adenylate cyclase-coupled bradykinin receptor in guinea pig ileum membranes. Therefore, the effects of various well-known bradykinin B2 receptor antagonists were examined at the level of bradykinin-induced inhibition of ileal adenylate cyclase activity and compared with both their binding affinities and their potencies to antagonize ileal contraction evoked by bradykinin. A group of three highly potent antagonists was found to be able to antagonize both bradykinin-induced adenylate cyclase inhibition and smooth muscle contraction. Several other antagonists abolished the bradykinin-induced ileal contraction but did not influence its action on adenylate cyclase. The compound [D-Nal1, Thi5,8, D-Phe7]bradykinin which is known to inhibit the bradykinin-induced contraction in the rat uterus but not in the guinea pig ileum was found to be a weak but selective antagonist for the adenylate cyclase-coupled bradykinin receptor in guinea pig ileum. Altogether, in guinea pig ileum membranes the inhibitory, adenylate cyclase-coupled bradykinin B2 receptor with pM affinity towards bradykinin exhibits a unique antagonist profile and is distinguished from the excitatory bradykinin B2 receptor with nM affinity towards bradykinin.

New photoaffinity labelled agonists of bradykinin.

Several photoaffinity labelled agonists of the peptide hormone bradykinin (BK) were synthesized by solid phase methods. Their biological activities and binding affinities were determined in both the isolated rat uterus (RUT) and guinea pig ileum (GPI). As photoreactive groups p-benzoyl-phenylalanine (Bpa) and the arylazides azidobenzoic acid (ABA) and azidosalicylic acid (ASA) were attached to the N-terminus of the BK agonists. In addition, Bpa was incorporated at different positions of the BK sequence. Three different types of BK agonists were used. Firstly, the photolabels ASA and ABA were attached to BK or to Lys-BK (kallidin). Secondly, tyrosine containing BK analogues, suitable for radioiodination, were labelled. This series is derived from the naturally occurring analogue phyllokinin [BK-Ile-Tyr(SO3H)] and from BK analogues with tyrosine at position 0 and 3. The third series includes several analogues with D-N-methyl-phenylalanine (D-NMe-Phe) at position 7, which selectively discriminate between the RUT and GPI bradykinin B2 receptors. Among the photoaffinity labelled BK agonists, the iodinatable Lys(ASA)-BK (50.8% on RUT, 73.0% on GPI), ASA-BK (26.3% on RUT), Bpa-BK-Ile-Tyr (13.6% on RUT, 14.0% on GPI) and the iodinated [D-Bpa-1, 3-I-Tyr0]-BK (15.5% on RUT, 19.0% on GPI) retained a relatively high biological activity compared with BK (100%). Thus, although BK agonists are known to allow only very restricted modifications without a strong reduction in biological activity, these compounds should be useful candidates for receptor labelling.

Potent photoaffinity labelled and iodinated antagonists of bradykinin.

Continuing the studies on photoaffinity labelled analogues of the peptide hormone bradykinin (BK), several labelled antagonists were synthesized and characterized regarding their biological activities on rat uterus (RUT) and guinea pig ileum (GPI). The photoreactive amino acid p-benzoyl-phenylalanine (Bpa) was incorporated in potent, iodinated BK analogues at positions -2, -1, 0 and 7. The newly synthesized BK antagonists were derived from HOE 140 ([DArg0, Hyp3, Thi5, D-Tic7, Oic8]-BK) or [D-Phe7]-BK. Because the application of Bpa requires an additional group for the introduction of 125I, iodinated tyrosine was inserted at different positions as a model for radioiodination. Suitable positions for incorporation of tyrosine residues are -1, 0, 3 and 7, whereas the compound with 3-I-Tyr at position 4 had only a low biological activity. The antagonists obtained by modification of HOE 140 generally retained a high antagonistic potency. In this group [D-Bpa-2, 3-I-D-Tyr-1, D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-BK (pA2 values 8.06 on RUT and 8.15 on GPI) and [Bpa-1, D-Arg0, 3-I-Tyr3, Thi5, D-Tic7, Oic8]-BK (pA2 values 7.55 on RUT and 8.07 on GPI) belong to the most active compounds. The incorporation of D-Bpa at position 7 also resulted in potent analogues. The antagonists [3-I-Tyr-1, D-Arg0, D-Bpa7]-BK (pA2 on RUT 7.69) and [3-I-Tyr-1, D-Arg0, D-Bpa7, Oic8]-BK (pA2 on GPI 7.53) are an alternative to the N-terminal modified HOE 140 analogues. Compounds with D-Bpa7 act as pure competitive antagonists, whereas the HOE 140 derivatives show a mixed antagonism. The comparison of the results between photoaffinity labelled agonists and antagonists suggests that modifications in the series of BK antagonists were better tolerated.

Bradykinin inhibits adenylate cyclase activity in guinea pig ileum membranes via a separate high-affinity bradykinin B2 receptor.

In guinea pig ileum membranes, the pre-stimulated adenylate cyclase activity was dose-dependently inhibited by picomolar concentrations of bradykinin exhibiting an apparent IC50 value of approximately 30 pM. At nanomolar bradykinin concentrations (> 1 nM) this effect was attenuated. The inhibition of ileal adenylate cyclase was completely prevented by both the bradykinin B2 receptor antagonist Hoe 140 (D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin) and pertussis toxin. The potency of bradykinin to inhibit ileal adenylate cyclase considerably correlates with a binding site with picomolar affinity for bradykinin. In addition, a second site was constantly found displaying nanomolar binding affinity for bradykinin. The occurrence of two independent bradykinin B2 receptors in guinea pig ileum membranes is further supported by three other lines of evidence: bradykinin stimulates [35S]GTP[S] (guanosine 5'-O-[3-thiotriphosphate]) binding to guinea pig ileum membranes in a biphasic manner with EC50 values which correspond to the affinities of both sites. In binding studies, the high-affinity site cannot be transformed into the low-affinity site in the presence of Gpp[NH]p (5'-guanylylimidodiphosphate). The specific binding of [3H]bradykinin to guinea pig ileum membranes was also biphasically inhibited by increasing concentrations of Gpp[NH]p. Thus, our results favour the existence of two separate bradykinin B2 receptors with different signal transduction pathways in guinea pig ileum membranes: one receptor with picomolar affinity for bradykinin which inhibits adenylate cyclase via a pertussis toxin-sensitive G protein of probably the Gi2 type and the other receptor with nanomolar affinity for bradykinin which might be responsible for bradykinin-induced stimulation of phosphoinositide hydrolysis.