Volume 382, Issue 1

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein ( KLK ) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes ( KLK4 - KLK15 ) within and adjacent to the original human KLK locus ( KLK1 -3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic ( KLK2-4 , KLK15 ) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

We have purified, cloned and characterized kallistatin, a tissue kallikrein-binding protein (KBP) in humans and rodents. Kallistatin is a unique serine proteinase inhibitor (serpin) with Phe-Phe residues at the P2 and P1 positions. Structural and functional analysis of kallistatin by site-directed mutagenesis and protein engineering indicate that wild-type kallistatin is selective for tissue kallikrein. Kallistatin is expressed and localized in endothelial and smooth muscle cells of blood vessels and has multiple roles in vascular function independent of the tissue kallikrein-kinin system. First, kallistatin induces vasorelaxation of isolated aortic rings and reduces renal perfusion pressure in isolated rat kidneys. Transgenic mice overexpressing rat kallistatin are hypotensive, and adenovirus-mediated gene delivery of human kallistatin attenuates blood pressure rise in spontaneously hypertensive rats. Second, kallistatin stimulates the proliferation and migration of vascular smooth muscle cells in vitro and neointima formation in balloon-injured rat arteries. Third, kallistatin inhibits the proliferation, migration and adhesion of endothelial cells in vitro and angiogenesis in the rat model of hindlimb ischemia. These results demonstrate novel roles of kallistatin in blood pressure regulation and vascular remodeling.

Muscarinic acetylcholine receptors in NG108-15 neuroblastoma x glioma cells, and ?-adrenergic or angiotensin II receptors in cortical astrocytes and/or ventricular myocytes, utilize the direct signaling pathway to ADP-ribosyl cyclase within cell membranes to produce cyclic ADP-ribose (cADPR) from ?-NAD + . This signal cascade is analogous to the previously established transduction pathways from bradykinin receptors to phospholipase C? and ?-adrenoceptors to adenylyl cyclase via G proteins. Upon receptor stimulation, the newlyformed cADPR may coordinately function to upregulate the release of Ca 2+ from the type II ryanodine receptors as well as to facilitate Ca 2+ influx through voltage-dependent Ca 2+ channels. cADPR interacts with FK506, an immunosuppressant, at FKBP12.6, FK506-bindingprotein, and calcineurin, or ryanodine receptors. cADPR also functions through activating calcineurin released from Akinase anchoring protein (AKAP79). Thus, some G q/11 coupled receptors can control cADPR-dependent modulation in Ca 2+ signaling.

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B 1 receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg 9 BK > desArg 9 BK >= LysBK>BK; apparent affinities of antagonists Lys[Leu 8 ]desArg 9 BK (pIC 50 8.4 ) > [Leu 8 ]desArg 9 BK (pIC 50 7.4)>>>HOE 140, a B 2 receptor antagonist, pIC 50 <5.0), and the B 2 receptor of the rabbit jugular vein (potency of agonists: LysBK = BK>>> LysdesArg 9 BK = desArg 9 BK and HOE 140 (pIC 50 9.0)>>>Lys[Leu 8 ]desArg 9 BK, pIC 50 <5.0). The second part describes speciesrelated B 1 receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg 9 BK >> desArg 9 B K and Lys[Leu 8 ]desArg 9 BK > [Leu 8 ]desArg 9 BK) and dog, rat, mouse and hamster B 1 receptors (desArg 9 BK = LysdesArg 9 BK and [Leu 8 ]desArg 9 BK = Lys[Leu 8 ]desArg 9 BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their Nterminus. The last part describes species-related B 2 receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B 2 receptors. Various hypotheses are considered to interpret these findings.

Bradykinin plays many roles in normal and pathological physiology, but rapid enzymatic degradation made elucidation of its functions extremely difficult. Development of effective degradation-resistant antagonists made it possible to delineate these roles and to open the way for development of new drugs to control pathology due to excess production of bradykinin. Presently available peptide bradykinin antagonists are extremely potent, are completely resistant to enzymatic degradation, and are orally available. Non-peptide bradykinin antagonists have also been discovered. Development of bradykinin antagonists as drugs for cancer, inflammation and trauma is anticipated.

Based on studies presented here and other published experiments performed with surviving tissue preparations, with transfected cells and with cells that constitutively express the human angiotensin I converting enzyme ACE and B 2 receptors, we concluded the following: ACE inhibitors and other endogenous peptides that react with the active site of ACE potentiate the effect of bradykinin and its ACE resistant peptide congeners on the B 2 receptor. They also resensitize receptors which had been desensitized by the agonist. ACE and bradykinin receptors have to be sterically close, possibly forming a heterodimer, for the ACE inhibitors to induce an allosteric modification on the receptor. When ACE inhibitors augment bradykinin effects, they reduce the phosphorylation of the B 2 receptor. The primary actions of bradykinin on the receptor are not affected by protein kinase C or phosphatase inhibitors, but the potentiation of bradykinin or the resensitization of the receptor by ACE inhibitors are abolished by the same inhibitors. The results with protein kinase C and phosphatase inhibitors indicate that another intermediate protein may be involved in the processes of signaling induced by ACE inhibitors, and that ACE inhibitors affect the signal transduction pathway triggered by bradykinin on the B 2 receptor.

Mitogenic signalling pathways from G protein-coupled receptors (GPCRs) to the mitogen-activated protein kinase (MAPK) cascade may involve ?- or ??-subunits of heterotrimeric G proteins, receptor or nonreceptor 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 B 2 receptor (B 2 R) 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 B 2 R and MAPK may exist than in COS-7 cells, suggesting a high degree of cellular specificity in mitogenic signalling.

Kinin effects on epithelial electrogenic ion transport are reviewed, with reference to the alimentary tract. The transported ion is usually chloride, but some epithelia also transport bicarbonate. The key components of the transport system are the sodium-potassium-chloride cotransporter, Na + -K + ATPase (both located basolaterally) and the CFTR chloride channel located apically). Activation of K + channels in both membranes may secondarily affect the anion transport mechanism. The types of kinin receptors that cause chloride secretion, the second messengers involved and the possible functional responsibilities of the kinin-activated secretory mechanism are discussed.

The role of the renal kallikrein-kinin system in the development of salt-sensitive hypertension was studied using mutant kininogen-deficient Brown-Norway Katholiek (BN-Ka) rats, which generate no kinin in their urine, and other hypertensive rat models. It was found that ingestion of a low sodium diet or infusion of NaCl in doses slightly above 0.15 M caused hypertension and sodium accumulation in erythrocytes and the cerebrospinal fluid of kininogen-deficient BN-Ka rats. Development of hypertension in the deoxycorticosterone-acetate-salt model was completely prevented by administration of a newly discovered inhibitor, ebelactone B, of carboxypeptidase Y-like exopeptidase (an urinary kininase). The urinary kallikrein excretion of spontaneously hypertensive rats was lower than that of Wistar Kyoto rats at 4 weeks of age and did not increase by administration of furosemide, a diuretic agent, although approximately 50% of the diuretic action of this agent was dependent upon the renal kallikrein-kinin system in normal rats. In conclusion, the renal kallikrein-kinin system works as a safety valve for excess sodium intake.

Cleavage of high molecular weight kininogen (HK) by plasma kallikrein results in a light chain and a heavy chain (HK). The light chain has two domains: D6, which binds (pre)kallikrein, and D5, which binds to anionic surfaces, including heparin as well as zinc. Initially, HK was thought to be important for surface-activated coagulation. HKa or D5 binds to the urokinase receptor on endothelial cells, thereby enhancing the conversion of prourokinase to urokinase by kallikrein, and, thus, cell-associated fibrinolysis. HKa or D5 is antiadhesive by competing with vitronectin binding to the urokinase receptor and/or forming a complex with vitronectin. D5 inhibits endothelial cell migration, proliferation, tube formation and angiogenesis, thus modulating inflammation and neovascularization.

Activation of the plasma kallikrein-kinin forming cascade takes place upon incubation with human umbilical vein endothelial cells. The mechanism by which initiation occurs is uncertain. Zinc-dependent binding of plasma proteins to gC1qR, cytokeratin 1, and perhaps u-PAR is requisite for activation to take place. We demonstrate here that during a 2 hour incubation time plasma deficient in either factor XII or high molecular weight kininogen (HK) fails to activate, as compared to normal plasma, but with more prolonged incubation, factor XII-deficient plasma gradually activates while HK-deficient plasma does not. Our data support both factor XII-dependent (rapid) and factor XII-independent (slow) mechanisms; the latter may require a cell-derived protease to activate prekallikrein and the presence of zinc ions and HK.

The kallikrein family of serine proteases has been investigated in many inflammatory disorders as molecular mapping, gene characterisation and cloning of kinin receptor genes have unfolded experimentally. In the molecular events of the inflammatory response the kallikrein cascade plays a significant role, since it is considered to initiate and maintain systemic inflammatory responses and immune-modulated disorders. A primary event is the chemotactic attraction of neutrophils which deliver the kallikrein-kinin cascade to sites of cellular injury and carcinogenic transformation of cells. The present study establishes the casual involvement of the kallikrein cascade in infection, inflammatory joint disease, acute transplant rejection, renal glomerular diseases, angiogenesis and carcinoma. We provide strong evidence for new or enhanced expression of kinin B1 receptors in inflammation, and additionally the induction of kallikrein genes in angiogenesis and carcinoma. The results provide insights into possible roles of kallikrein inhibitors and kinin receptor antagonists.

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit µ- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and ?L110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefinB by the corresponding KD2 sequence resulted in a calpain inhibitor of K I = 188 nM. Deletion of L110, which forms a ?-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of µ-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substratelike cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatintype inhibitors do not bind to the calmodulinlike domain of the small subunit of calpain, and their interaction with the µ-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatinlike mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.

We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and nonfluorogenic peptides based on the Bauhinia inhibitors` reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp ( K m 1.42 M, k cat 0.06 s -1 , and k / K 4.23 x 10 4 M -1 s -1 ), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence ( K m 0.43M, k cat 0.00017 s -1 , and k / K 3.9 x 10 2 M -1 s -1 ). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with K I values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

A novel secreted aromatic amino acid decarboxylase-like molecule was identified in the excretory/secretory products of L3/L4 larvae as well as in an extract of adult Dirofilaria immitis . The secretion of the enzyme was developmentally regulated. Peak enzyme activities were detected in the culture medium before and after the molting of L3 larvae in vitro . The enzyme was purified from D. immitis adult extracts and the excretory/secretory products of L3/L4 larvae using different chromatographic methods followed by isoelectric focusing and SDS-PAGE. The enzyme has a molecular mass of 48 kDa and a pI of 5.6, and shows a specific enzymatic activity towards the aromatic amino acid substrates phenylalanine, tyrosine and tryptophan. The enzymes activity did not show an absolute requirement for exogenous pyridoxal-5-phosphate. However, addition of pyridoxal-5-phosphate at 5 μM in the reaction increased the enzyme activity greatly. The enzyme had the ability to catalyze the formation of dopamine from L -dopa. Studies on the effects of inhibitors on the enzyme activity showed that the enzyme was sensitive to Pefabloc and p -chloromercuribenzoic acid, but not to diisopropyl flurophosphate. The K m values of the enzyme for H-Phe-AMC, H-Tyr-AMC and H-Trp-AMC were calculated to be 32.1 μM, 35.1 μM and 29.1 μM, respectively.

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the Nterminal Lys residue of [desArg 10 ]Lysbradykinin. Significantly, the B1 receptor antagonist [desArg 9 ,Leu 8 ]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the classical pharmacological properties of this receptor subtype.

The kinin B 1 receptors (B 1 Rs), stimulated by desArg 9 kinins, are completely inducible, notably following treatment of animals with bacterial lipopolysaccharide. Several studies based on cultured cells have suggested a form of autoregulation of kinin receptors, because B 2 receptor (B 2 R) or B 1 R stimulation could transcriptionally upregulate B 1 R expression. The B 2 R may rather be downregulated in inflammatory conditions. A rabbit B 2 R-green fluorescent protein GFP) conjugate stably expressed in HEK 293 cells was rapidly internalized in response to the agonist bradykinin. Ligand-induced receptor cycling was documented applying confocal microscopy. The results confirm agonist-induced B 2 R endocytosis, but extensive recycling to the cell membrane does not support agonist-induced downregulation.

Sphingosine kinase phosphorylates sphingosine to generate sphingosine 1-phosphate, a phospholipid that has been implicated in signaling by a number of transmembrane receptors and was recently shown to act as a ligand for a specific class of G protein-coupled receptors. Here we show that the G protein-coupled bradykinin B 2 receptor activates sphingosine kinase leading to a time- and dose-dependent elevation of cellular sphingosine 1-phosphate levels that was blocked by the sphingosine kinase inhibitor dihydrosphingosine. Furthermore, increasing doses of this inhibitor partially affected the bradykinin-mediated ERK/MAP kinase activation and fully blocked the protein kinase C-independent component of the signaling pathway from the B 2 receptor to the ERK/MAP kinase cascade. Overexpression of sphingosine kinase did not additionally increase the bradykinin-induced ERK/MAP kinase activity, indicating a permissive rather than activating role of sphingosine 1-phosphate in B 2 receptor-mediated mitogenic signaling.