J Pharmacol Sci 93, 1 – 20 (2003) Journal of Pharmacological Sciences ©2003 The Japanese Pharmacological Society Critical Review

Roles for the Kallikrein- System in Inflammatory Exudation and Pain: Lessons From Studies on -Deficient Rats Akinori Ueno1,# and Sachiko Oh-ishi2,*

1Department of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan 2Basic Research Division, Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan

Received May 27, 2003

Abstract. Roles for the kallikrein-kinin system in inflammation have been investigated exten- sively, and many reviews on this topic have been published during the 50 years since the dis- covery of in 1949. Recent progress in the field has been remarkable with the help of experiments using gene-targetted transgenic or knockout mice, which have added further valu- able information in addition to previous results obtained from pharmacological and biochemical studies using purified and isolated components of the system. Furthermore, much knowledge has been accumulated as a result of the development of various bradykinin agonists and antagonists. In this review, we focused on the data obtained from the kininogen-deficient rat, which is a natural mutant, and discuss the results in comparison with those from bradykinin receptor knock- out mice. These data have clarified that endogenous bradykinin exerts a most important role in inflammatory exudation along with prostanoids, preferentially to , serotonin, or neuro- peptides. In inflammatory pain perception also, bradykinin produced in the local perivascular spaces stimulates polymodal pain receptors in conjunction with co-helpers such as prostanoids, vanilloids, and neuropeptides. These important roles are concluded based on consistent results obtained from experiments using several antagonists of bradykinin, kininogen-deficient rats, and bradykinin receptor knockout mice.

Keywords: kallikrein-kinin system, bradykinin, paw edema, pleurisy, inflammatory exudation

1. Introduction from the kidney instead of from the pancreas (kallikreas) (4). The kallikrein-kinin system represents an endogenous Bradykinin, an active peptide produced by the kalli- enzyme cascade, the triggering of which results in the krein-kinin system, was first reported by Rocha e Silva activation of kallikreins to produce the active peptide et al. in 1949 (5) as a substance, produced when human bradykinin, a molecule that exerts various potent bio- plasma was mixed with venom of the snake Bothlops logical effects, including hypotension, microvascular jararaca, that slowly (brady) contracted (kinos) the iso- permeability increase, contraction, and lated ileum. Bradykinin was also produced in human inflammatory pain production (1, 2). Historically, plasma kept in a glass tube and acted as a pain-producing kallikrein had been first described as a hypotensive substance, as described by Keele and Armstrong (6). substance found in urine by Frey et al. in the 1930’s as In fact, bradykinin when injected into experimental described in Ref. 3, although later, the kallikrein found animals can cause all 4 signs of inflammation, that is, in urine was identified as a glandular kallikrein derived swelling, heat, redness, and pain, which had been docu-

# mented from the era of ancient Greece; and so brady- Present address: DDS Institute, The Jikei University School of Med- kinin has been considered to be one of the important icine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo 108-8461, Japan *Corresponding author. FAX: +81-3-5791-6120 mediators of inflammation (7). E-mail: [email protected] During the period of 1955 – 1970, congenital defi- ciencies in various plasma proteases, substrates, and Invited article

1 2 A Ueno and S Oh-ishi inhibitors, including plasma prekallikrein or , secreted into the circulation and then distributed to all were discovered; and mechanism of activation of the of the tissues in the body (18). As indicated in Fig. 1A, blood coagulation cascade as well as the plasma prekallikrein is secreted and present in the blood as a kallirkein-kinin system were extensively studied in vitro pro-enzyme, and it is activated to plasma kallikrein by by using plasma from affected individuals (8). In 1979, the action of active Factor XII, that is, XIIa; and in turn, the first case of experimental animals with a genetic plasma kallikrein cleaves H-kininogen to release brady- deficiency of kininogens, components of the kalllikrein- kinin (2, 19). kinin system, was discovered (9, 10), thus affording a However, other kinin-producing enzymes (kinino- tool for in vivo experiments. The data obtained from genases), known as glandular kallikreins, are synthe- these kininogen-deficient rats allowed extensive sized in the glandular tissues and are found in their progress to clarify the in vivo roles of the kallikrein- active forms in various tissues and their fluids, including kinin system. Further progress was made by recently the pancreas, pancreatic juice, salivary glands, saliva, obtained knowledge from kinin-receptor gene-disrupted kidneys, urine, or sweat (4, 20). Glandular (or tissue) mice (11, 12), which is mostly in accord with the results kallikreins are different enzymes from plasma kallikrein obtained from the in vivo experiments with various with respect to their enzymatic characterization, genes, antagonists for bradykinin receptors (13, 14). This and amino acid sequences (21). Therefore, the kalli- review focuses on the results obtained from kininogen- krein-kinin system comprises 2 enzyme systems, that is, deficient rats and discusses the in vivo significance of the plasma kallikrein-kinin system and the glandular (or the activation of the kallikrein-kinin system in light of tissue) kallikrein-kinin system, each of which may play the information obtained from the bradykinin receptor- different physio-pathological functional roles (Fig. 1). knockout mice as well as from the use of bradykinin B1 The initial activation of the plasma kallikrein-kinin and B2 receptor antagonists. system starts from the negatively charged surface-acti- vation of Factor XII, and this activation also puts the 2. Components of the kallikrein-kinin system in intrinsic blood coagulation cascade into action (19). As plasma Factor XII and prekallikrein are present in the blood as pro-enzymes, they must be activated for both cascades First of all, characteristics of the kallikrein-kinin to start. Therefore, during the decates of 1960’s and system may be summarized by a slight glance of the 1970’s, the search for the mechanism of the initial historical events of the field; and some references activation of both systems was extensively undertaken, describing purification, cloning, and analysis of the and much progress resulted from experiments using components as well as congenital deficiencies of some congenitally factor-deficient plasmas, which had been components are cited. Most components in the kallikrein- discovered one after another during these decades (re- kinin system of mammals including humans are present viewed by several authors (1, 2, 19)). in the plasma, such as Factor XII (15), prekallikrein A deficiency of Factor XII was first described by (16), high-molecular-weight (H)-kininogen (17), and Ratnoff and Colopy in 1955 as that of Hageman factor low-molecular-weight (L)-kininogen (17). These pro- (22), because a factor necessary to start intrinsic blood teins are known to be synthesized by the liver and coagulation was lacking in Mr. Hageman’s blood

Fig. 1. The kallikrein-kinin system. The kallikrein-kinin system comprises of the plasma kallikrein-kinin system (A) and the glandular (or tissue) kallikrein- kinin system (B). In panel A, initiation of Factor XII-activation occurs when plasma is in contact with negative sur- faces, such as kaolin, glass tubes, skins, etc. Prekallikrein and H-kininogen also function as cofactors for the activation of Factor XII. Several differences in the plasma (A) and the glandular (B) kal- likrein-kinin systems were found, such as in vivo roles, the enzyme profiles, and the product , bradykinin and , respectively. Inflammatory Role for the Kinin System 3

(Fig. 2) (23). The kaolin-activated thromboplastin time In addition to the above-described activities, a new (APTT) of Mr. Hageman’s plasma was significantly role for H-kininogen was found: its heavy chain contains prolonged, that is, around several min versus 50 – 70 s 2 conserved moieties of the sequence (-Gln-Val-Val- for citrated normal human plasma, which made certain Ala-Gly-) that act as inhibitors of SH-proteases, such as Factor XII that was an initiating factor for the intrinsic papain and bromelain (32). These repeated sequences blood coagulation cascade (23). Initiation of the plasma reside in all kininogens, including T-kininogen. How- kallikrein-kinin system was also proved to occur by a ever, the in vivo significance of these inhibitors has negative surface-mediated activation of Factor XII, not yet been clarified. Furthermore, T-kininogen was because kaolin-activation of Factor XII-deficient plasma discovered only in rat plasma (33 – 35), where its level could not yield bradykinin (8, 19). increased when rats received an inflammatory stimulus Moreover, prelkallikrein-deficient plasma (24) and or bore cancer. T-kinin (Ile-Ser-bradykinin, Fig. 3) is H-kininogen-deficient plasma (25 – 28) showed a pro- produced from T-kininogen by the action of trypsin in longed APTT and decreased or failed kinin formation high concentration (35), and at the moment, no endo- when brought into contact with kaolin or glass. There genous enzyme to cleave T-kinin is known. The physio- have been no reports of any bleeding tendency or related logical role for T-kininogen, an acute phase protein, is disorders in patients deficient in Factor XII, pre- yet unknown. The gene for T-kininogen is different kallikrein, or kininogens; in fact, it was reported that from H- or L-kininogen (33), but T-kinin acts on the Mr. Hageman died of pulmonary thrombosis (29). B2 receptor to cause a similar effect as bradykinin (35). However, there have been conflicting reports on throm- Based on the accumulated information about the botic tendency in Factor XII deficiency. Some reports activation cascade, the plasma kallikrein-kinin system is showed an increased incidence of thrombotic complica- now widely recognized as the scheme illustrated in tions in patients with Factor XII deficiency because of Fig. 1. Factor XII in the blood or interstitial fluids is less activity of Factor XII-dependent plasmin production activated upon contact with negatively charged surfaces (30), but others reported that there was no difference in to yield its active form, XIIa, by cleavage with enzymes the thromboembolic incidence between normal and such as plasma kallikrein. A key enzyme, XIIa cleaves Factor XII-deficient patients (31).

Fig. 2. A photograph of Mr. Hageman, cited from Professor Ratnoff’s report. The first case of Factor XII deficiency. Mr. John Hageman, a freight switchman on the New York Central Railroad, had no bleeding tendency and received an operation to cure his Fig. 3. Amino acid sequence around kinins in the kininogens of peptic ulcer. The first initiating factor for intrinsic clotting of plasma several mammals, including humans. Cleavage sites recognized by when it comes in contact with glass was missing in Mr. Hageman’s plasma and glandular kallikreins are shown by arrows. Bradykinin 9 plasma. So Professor Ratnoff named this factor “Hageman factor”, and T-kinin act on the B2 receptor. Des-Arg -bradykinin and des- and its absence was designated as “Hageman trait”. Photograph, Arg10-kallidin are produced by the action of kininase I, and they act reprinted from Ref. 23 with permission. on the B1 receptor. 4 A Ueno and S Oh-ishi prekallikrein and Factor XI to start the cascades of the found to be immediately converted to bradykinin by kallikrein-kinin system and intrinsic blood coagulation. aminopeptidases in the circulation (4). Cleavage of Then, plasma kallikrein cleaves prefentially H-kinino- bradykinin from kininogens has been also extensively gen, to release bradykinin (8, 19). The complex nature studied, as shown in Fig. 3, where the amino acid of the activation of Factor XII and prekallikrein have sequences in the vicinity of bradykinin in various been extensively studied by using reconstruction mix- animals are shown. Human urinary kallikrein cleaves tures of these isolated components (8, 15, 16, 19). It human L-kininogen to yield kallidin, that is, Lys-brady- was found that prekallikrein circulates in the blood as kinin (4), whereas plasma kallikrein yields bradykinin a complex with H-kininogen (36), which may provide preferentially from H-kininogen (8). These cleavages a conformational susceptibility of H-kininogen to the are the same in the case of bovine and equine glandular enzymatic action of plasma kallikrein and also may give kallikrein (4, 20) and plasma kallikrein as those in prekallikrein a longer biological life in the blood stream humans. However, glandular kallikrein from rats or (36). Cleavage of Factor XII to XIIa by endogenous mice produces bradykinin from L-kininogen, and their enzymes such as prekallikrein or plasma kallikrein was plasma kallikrein also produces bradykinin from H- also evaluated (8, 19, 37). Then, questions arose as to kininogen (16). Interestingly, hog pancreas kallikrein what would first activate prekallikrein or Factor XII, and does not produce kinins from rat H-kininogen (41). what are negative surfaces in a living body and how do However, the kininogenase from the snake venom they make contact with each other in the circulating cleaves bradykinin from bovine and human H- or L- blood. To answer these questions, many studies on kininogens. These facts indicate a species difference in reconstruction of systems with purified factors have the action of kallikreins on substrates of other animal been extensively undertaken, but in these experiments species. slight contaminants have always been problematic. Receptors for bradykinin have been classified as B1 Researchers have been seeking animals deficient in and B2 kinin receptors (42, 43), and they are autacoid these factors, which may afford in vivo tools. Scattered receptors having 7 transmembrane moieties coupled to reports on animals deficient in Factor XII, that is, dogs G-protein (43). Characteristics of B1 and B2 receptors (38, 39), or in prekallikrein, that is, horses (40), have and their agonists and antagonists have been reviewed appeared; however, no subsequent information was by several authors (13, 14). Classical activities caused made available. Although our knowledge of the bio- by bradykinin are due to the B2 receptor, which is chemistry of the kallikrein-kinin sytem has made signifi- constitutively expressed in every organ or tissue, cant progress, obtained by the use of factor-deficient whereas the B1 receptor is induced after exposure to plasmas, the following questions have not yet been endotoxin or some inflammatory stimuli (44). Research answered in spite of the extensive studies on systems into how the B1-receptor is induced has continued, but reconstructed with purified isolated components: 1) Is it is a mystery why the isolated aorta of rabbits and the plasma kallikrein-kinin system preferentially in- isolated rat ileum gradually expresses the B1 receptor, volved in inflammatory reactions? 2) Is the glandular that is, gradually becoming contractible by des-Arg9- kallikrein-kinin system related to the blood pressure bradykinin (43, 45). Selective antagonists for B1 and B2 control along with the renin- system? Since receptors have been developed and reviewed (13, 14). urinary kallikrein had been discovered because of its hypotensive effect of urine (3), initial studies on the 3. Discovery and characterization of congenital kallikrein-kinin system had been focused on the cardio- kininogen-deficient Brown Norway-Katholiek rats vascular roles of bradykinin. In fact, bradykinin shows a potent hypotensive effect in various experimental In 1979 and 1980, Damas and Adam reported that animals as well as in humans; however, its potent effects a rat strain, Brown Norway mai, showed no kinin on the microvasculature, that is, causing a vascular formation and low kallikrein activity in their plasma permeability increase, and on pain production shifted even when activated with kaolin (9, 10). This was the attention to its role as an inflammatory mediator (1, 2). first report of a congenital deficiency of a component of There are some reported species differences in the the kallikrein-kinin system in an experimental animal. purified isolated components in this system, and so there However, the same rat strain, Brown Norway (B/N should be more in vivo studies for identification of the mai pdf f), in a farm of Kitasato University expressed specific role of the activation of the system in each normal APTT and kinin formation (46). The latter rats species. For example, glandular kallikrein produces had been originally introduced from Microbiological kallidin (Lys-bradykinin) in humans, cows, and horses, Associates, USA to the National Institute of Health of preferentially from L-kininogen (4, 20); and kallidin was Japan during the 1950’s – 60’s, and they were bred at Inflammatory Role for the Kinin System 5 the Medical School of Kitasato University by T. Nakano (46). Later, Pisano’s group at NIH in the USA examined (46). Then kininogen-deficient B/N rats were sent the B/N rats at the NIH farm, and they reported that the from the rat colony at Katholiek University, Leuven, B/N rats at that farm showed normal kinin formation Belgium, to Kitasato University in 1981; and since then, (48). Damas mentioned that their “B/N-Katholiek” rats they have been bred by Nakano and his colleagues. The had been obtained from the NIH long before, but he result of Damas and Adam (9, 10) was confirmed in that did not know the cause for the deficiency. The origin B/N rats from Katholiek University showed no kinin of Brown Norway rats in the USA is documented as formation by kaolin activation, no kininogen, and less follows: Dr. H.D. King of the Wistar Institute captured plasma kallikrein activity; and the plasma of the B/N some wild Norway rats (Rattus norvicans) in the vicinity rats did not correct the prolonged APTT of kininogen- of Philadelphia and proceeded to produce an inbred deficient human plasma, such as plasma from patients strain of them, 35 generations by 1934, which is known with the Fitzgerald or Fujiwara trait (47). So, to differen- as the Brown Norway rat (49, 50). tiate those rats, we named the kininogen-deficient rat B/N-Ka rats did not show any physiological differ- strain B/N-Katholiek (B/N-Ka) and the normal strain ence from B/N-Kit rats except that they showed pro- from Kitasato University as B/N-Kitasato (B/N-Ki) longed APTT and did not release bradykinin by the

Fig. 4. Growth curves, body weight changes during life, and average litter size of B/N-Katholiek (kininogen-deficient rats from Katholiek University, Belgium), B/N-Kitasato (normal rats from Kitasato University), and B/N-Sea (normal rats from Seiwa Experimental Animal Co.). These rats were bred in the experimental animal laboratory of Kitasato University, and data were obtained during the period of 1987 – 1997. Photos show male B/N-Kitasato (Ki) rats (10-week-old), and a mating couple of B/N-Katholiek (Ka) and their babies (2 – 3 days). No difference was seen between the appearance of these rats. Part of the data is from Ref. 55. 6 A Ueno and S Oh-ishi action of kaolin activation or by urinary kallikrein (51). was Mendelian autosomal recessive (53), and it was Their growth curve showed no difference from that of associated with a low level of prekallikrein with the B/N-Ki rats and even commercialy available normal same profile as reported for human H-kininogen defi- B/N-rats (B/N-Sea) from Seiwa Experimetal Animal ciency (25 – 27). They showed a marked difference Co. (Fukuoka) as shown in Fig. 4, although the body only when they received an inflammatory stimulus or weights of all these Brown Norway strain rats were invasion, for example, paw edema or pleurisy induced significantly less than those of Wistar and Sprague- by carrageenin, as shown in the next section. It was also Dawley (SD) rats at the corresponding ages (52). The discovered that B/N-Ka rats produced hypertension in kininogen deficiency did not influence fertilization, response to DOCA-salt treatment faster than B/N-Ki pregnancy, breeding, term for contraception, or litter animals, and this hypertension was associated with the size as compared with normal B/N-Ki rats when the absence of urinary kinin (54); thus bradykinin may pre- B/N-Ka rats were kept and bred under the same condi- vent to produce hypertension in unknown mechanism tions (52). The litter size of all the 3 Brown Norway (54). Urinary kinin secretion in ureter urine was mostly strain rats is significantly smaller (about 5) than that of null in B/N-Ka rats when assessed by EIA for kinin, SD rats (about 18 – 20), as shown in the table in Fig. 4. while bradykinin was found in normal B/N-Ki urine The mode of inheritance of the kininogen deficiency (55). Furthermore, during intravenous infusion of

Fig. 5. High-molecular-weight-kininogen (HK) contents in plasma (A) and liver microsomes (B), and secretion of kininogens, HK (C) and T-kininogen (TK) (D), from primary cultures of the hepatocytes of B/N-Kitasato (Ki) and B/N-Katholiek (Ka) rats. Contents of HK and TK were measured by RIA using antibody to the light-chain of HK and monoclonal antibody to TK, respectively; and the values are expressed as means with S.E.M. Hepatocytes were isolated from B/N-Ki and B/N-Ka rats, and cultured for 48 h. * and ** indicates statistically significant difference from the corresponding data of Ki at P0.05 and P0.01, respectively. Details are described in Ref. 57. Inflammatory Role for the Kinin System 7 purified rat L-kininogen into B/N-Ka rats, bradykinin was detected in the ureter urine, but a small amount of kinin was found when H-kininogen was infused (56). Therefore, this experiment proved that rat urinary kinin is derived from L-kininogen and not from H-kininogen (56). A tissue distribution of H- or L-kininogen was assumed, as they were transported to the interstitial fluid by the blood supply; and therefore, most of the tissue contents paralelled the blood contents. However, the liver (Fig. 5B) and the kidneys of B/N-Ka contained immunoreactive kininogen at about 60% of that in B/N- Ki (57, 58); and so it may be assumed that kininogens are synthesized. It was found that the plasma H- and L- kininogen deficiency was caused by defective kininogen secretion by the liver (57, 59). Biosynthesis of kinino- gens was studied by using pulse and chase experiments with 35S-methionine for primary cultures of hepatocytes to detect immunochemically H- and L-kininogens of B/N-Ka and B/N-Ki rats (59). H- and L-kininogens synthesized de novo by the liver were cellular forms, showing larger molecular sizes (108 and 77 kDa, respec- tively) than the released forms in the plasma (100 and 66 kDa, respectively); and they are processed to be mature forms and then transported for secretion into the blood stream. In the primary cultures, kininogens of B/N-Ka were synthesized by the liver in similar sizes as the normal ones, but retained in the liver microsomes and not released into the medium (Fig. 5C). Sequences for genomic DNAs of H- and L-kininogen of Wistar rats were reported (33), and both kininogens were concluded to be derived from the same gene and produced by alternative splicing. Complementary DNAs of H-kininogen for B/N-Ka and B/N-Ki rats were Fig. 6. Gene targetted construction of control and mutated DNA, analyzed; and a point mutation of G to A at nucleotide and H-kininogen secretion from transfected COS-1 cells. Expression 487 was found in the cDNA of B/N-Ka rats, providing of HK mRNA in the transfected COS-1 cells was examined by a change from alanine 163 to threonine, which was Northern blot analysis (A) and reverse transcription PCR (B). COS-1 the only mutation in the entire coding region (60). To cells transfected with each constructed plasmid containing G or A (amino acid: alanine or threonine) at nucleotide position 487 examine if this mutation is the cause of defective (plasmids 1 to 6) were incubated for 24 h, and contents of HK in secretion of kininogens, nucleotide fragments containing the cell homogenates (C) and the supernatant (D) were measured the mutation or normal sequence were constructed; and by RIA. Samples (1 to 6) were prepared from cells transfected both B/N-Ki and B/N-Ka rat cDNA fragments were with the plasmids constructed from the following cDNAs: the normal / HK cDNA (pHKKI, lane 1); the deficient HK cDNA (pHKKA, introduced into a vector, pRc CMV, for construction lane 2); the mutated HK cDNA with one-point mutation of G to A of expression plasmids, which were used to transfect at nucleotide 487 constructed by PCR (pHKM, lane 3); the normal COS-1 cells. These transfected cells were examined for HK cDNA bearing the 921-bp BglII-SphI fragment (nucleotide secretion of kininogens in vitro. As shown in Fig. 6, after residue 371 – 1291) of the deficient HK cDNA (pHKNM, lane 4); the deficient HK cDNA bearing the 921-bp BglII-SphI fragment 24 h in culture, the COS-1 cells transfected with the (nucleatide residues 371 – 1291) of the normal HK cDNA (pHKMN, mutated fragment A showed a lower concentration of lane 5); and the control plasmid (pRC/CMV, lane 6). Cells tranfected H-kininogen in the medium than those containing the with the plasmids containing mutant nucleotide A (lanes 2 to 4) normal base of G; and in contrast, cellular contents of showed lower secretion and higher intracellular content of HK than those of cells transfected with plasmids containing normal G (lanes 1 the kininogen were higher in the cells with threonine 163 and 5). Thus, the result indicates that the one-point mutation at than alanine (60). These results strongly indicate that nucleotide position 487 (G to A) is responsible to defective secretion only 1 amino acid mutation (alanine to threonine) in of HK. Reproduced from Ref. 60 with permission. 8 A Ueno and S Oh-ishi the heavy chain of kininogens of B/N-Ka rat causes vity of H-kininogen. As the responsible amino acid defective secretion of H-kininogen and L-kininogen by sequences for SH-protease inhibitor reside in the heavy the liver. chain at positions 188 to 192 and 310 to 314 in the Before evaluating the role of kinin using kininogen- case of human kininogens (32, 33), a mutation of alanine deficient rats, it may be necessary to make careful and to threonine at 163 in the kininogens of B/N-Katholiek precise examination of phenotypes for the kininogen- may not be expected to affect the activity. Moreover, deficiency of this animal species (61). The results of the part of the sequence of H-kininogen for attachment precise examination of the contents of the components to prekallikrein is reported to be present in the L-chain of the kallikrein-kinin system, including H- and L- of H-kininogen (19, 33), and so it may not be either kininogens, in the plasma of kininogen-deficient B/N- affected by the mutation. Furthermore, reaction of B/N- Ka rats are illustrated in Fig. 5. Plasma levels of H- Ka rats in response to exogenous bradykinin, such as kininogen in the B/N-Ka rats were only 3% that of the blood pressure change or contraction of isolated ileum, normal B/N-Ki rats as detected by radioimmunoassay etc., was not different from that of normal B/N-Ki. using antibody to its L-chain. However, the T-kininogen level and its increasing feature in inflammatory stimuli 4. Role of the kallikrein-kinin system in inflam- were not different from those of normal B/N-Ki rats matory exudate formation (57, 61). The content of H-kininogen in the liver of B/N-Ka rats was about 60% of the normal level. Swelling is one of the signs of inflammation and it is Although B/N-Ka liver could not secrete produced H- a resultant phenomenon of vascular permeability in- and L-kininogens, it could secrete normally T-kininogen crease in the microvasculature; that is, exudate forma- (Fig. 5D). These results suggest that mutated kininogen tion at inflammatory sites caused plasma to leak into the of the B/N-Ka rats can not be secreted by the liver, interstitial space of the concerned tissues to make its but may be processed by the liver in the lysosomes. volume expand. Bradykinin expresses potent activity for The mutated H-kininogen isolated from the liver increasing vascular permeability in the injection site, microsomes of B/N-Ka rats showed normal clotting when assessed by the dye leakage method, as shown in activity and SH-protease inhibitor activity; and more- Fig. 7 (62 – 64). Bradykinin, as well as other agonists, over, this H-kininogen produced bradykinin by the such as histamine, 5-hydroxytryptamine (5-HT), and action of rat plasma kallikrein or urinary kallikrein (57). platelet-activating factor (PAF), when intradermally These results provided some information for analyzing injected into dorsal skins of mice, rats, guinea pigs, and the important amino acid sequences for functional acti- rabbits, causes plasma exudation at the site. Animals

Fig. 7. Vascular permeability increase induced by several agonists, including bradykinin, in the skins of mouse, rat, guinea pig, and rabbit when assessed by dye exudation. Animals were pretreated with an intravenous injection of 50 mg/kg pontamine sky blue at 10 min before the agonist injection. Exudation was assessed by measurement of the dye amount extracted from the leakage site in the skin where the agonists had been intradermally injected. Agonists used were platelet-activating factor (PAF), bradykinin (BK), 5-hydroxytryptamine (5-HT), and histamine (Hist) at the indicated dose (nmol) per site in the dorsal skin of each animal. The animals were killed by exsanguination 50 min after the agonist injection, and dye was extracted and measured as described in Refs. 62 – 64. Inflammatory Role for the Kinin System 9 intravenously injected with pontamine sky blue dye, when stored in a glass tube (6). which binds to serum albumin, were used to assess the The production of these permeability factors in the activity of agonists to form plasma exudates. The dye, serum was studied in vitro during the 1970’s – 1980’s which had passed through the vascular endothelial cells to clarify the mechanism of activation of the kallikrein- together with plasma protein, was extracted from each kinin system by using plasma deficient in various site. As seen in Fig. 7, when comparing the dose- components of the kallikrein-kinin system. For example, response curves for agonists tested among the species, the factors produced in glass tubes containing various bradykinin showed mostly similar potencies among the dilutions of human plasma were measured in terms of animals tested, whereas PAF showed great variation esterase activity by using a synthetic substrate, such as among the species,with the least activity being shown in H3-arginyl methyl ester (TAME), and compared with the the rabbit. These results indicate that exogenously permeability factors previously assessed in vivo (cf., administered bradykinin shows mostly similar potency Fig. 8: A and B (68)). These data expressed a parallel in the various experimental animal species commonly relationship between PF/Nat and direct esterase activity used, although an amount of endogenously produced and between PF/Dil and prekallikrein-activating acti- bradykinin depends on the balance of kinin production vity. That is, esterase activity of normal human plasma (kininogenase activity) and degradation (kininase acti- undiluted and at low dilution, estimated by TAME vity) in each animal species. Therefore to evaluate the in hydrolysis, corresponded to PF/Nat; and prekallikrein vivo role of endogenous bradykinin, one should account activator activity, assayed as esterase activity produced for the biological life of bradykinin by examining its in a mixture of human plasma at various dilutions biosynthesis and metabolism. Since the in vivo life of stored in glass tubes and isolated prekallikrein, showed a bradykinin in normal animals is so short, being less peak at the dilution of 200 times and accorded with the than 15 s in the circulation, it is reasonable to consider profile for PF/Dil. In Fig. 8C, prekallikrein activator that bradykinin is produced locally and acts as a local activity in various human plasma samples deficient in hormone. the components indicated is shown. Factor XII-deficient In 1953, Mackay et al. reported globulin permeability plasma did not induce prekallikrein activator activity, factors, PF/Age (a permeability factor produced after and both Fletcher plasma (prekallikrein-deficient storage of guinea pig serum for several days at 2LC) plasma) and Fitzgerald plasma (H-kininogen-deficient and PF/Dil (a permeability factor that was produced in plasma) showed low activities. Therefore it was proved diluted serum stored in glass tubes), and these factors that the production of prekallikrein activator activity induced dye exudation in the guinea pig skin where they (possibly active Factor XII, i.e., XIIa) to result in the had been injected (65). The production of PF/Dil was complete production of endogenous bradykinin at the inhibited in the presence of soya bean trypsin inhibitor inflammatory site needed the presence of Factor XII, (SBTI), which is known as an inhibitor for plasma H-kininogen, and prekallikrein and that the lack of the kallikrein, and also inhibited by some other inhibitors any one of them resulted in a defective vascular perme- present in undiluted plasma, because dilution led to ability increase; these findings are in line with the patho- higher activity; and the highest activity (peak) was found logical bradykinin-induced increase in vascular perme- at 200-fold dilution (Fig. 8A). Later PF/Nat, perme- ability. ability factor Native, was used instead of PF/Age, Furthermore, as seen in Fig. 8C, among the various since fresh rabbit serum contained active PF similar to deficient plasmas the most prominent prekallikrein that in undiluted human serum (66). The endogenous activator activity was found in the plasma of a HANE permeability factor was also produced in human plasma, (hereditary angioneurotic edema) patient who had a and it was suggested to be a mediator in mild thermal deficiency of C1 esterase inhibitor (C1iNH) (69, 70). injury, because the dye exudation in the animal skin HANE attacks have been documented as a symptom of of thermal injury was inhibited by SBTI, but not local edema, and the involvement of the second compo- susceptible to antihistamine (66). Spector reviewed nent of complement and bradykinin was suggested (71) the substances that induce a permeability increase, and because C1iNH is a potent inhibitor for Factor XIIa mentioned that bradykinin and kallidin were as potent and plasma kallikrein (70). These characteristics well as histamine and 5-HT (67). This phenomenon of explain the highest prekallikrein activator activity in production of permeability factors by a contact acti- HANE plasma (Fig. 8C). Angioedema attacks in patients vation of serum of humans, rats, and rabbits stored in with C1iNH deficiency are strongly suspected to be glass tubes attracted considerable attention in light of caused by endogenous bradykinin, but no in vivo evi- the initiation of the blood coagulation system (19) and dence has yet been reported. Recently C1iNH knockout the production of pain-inducing factor in human serum mice were produced by the targetted disruption of the 10 A Ueno and S Oh-ishi

C1iNH gene (72). It was reported that these C1iNH- These data strongly suggest that bradykinin causes disrupted mice showed a tendency to express increased exudation and edema attacks in HANE patients through vascular permeability as indicated by a blue nose and the bradykinin B2 receptor. blue ears when Evans blue dye was injected to compare Other examples for bradykinin as a major mediator of them with wild type mice, and this permeability increase vascular permeability increase in inflammation are the was reversed by treatment with human C1iNH. Dye following: In vivo experiments performed using kinino- exudation in the C1iNH-deficient mice was also sup- gen-deficient rats, B/N-Ka, in comparison with normal pressed by injection with a bradykinin B2-receptor B/N-Ki rats, clearly indicated that kinin produced from antagonist, Hoe 140, and enhanced by a kininase II H-kininogen could be a main mediator for exudation inhibitor, captopril. Furthermore, the vascular perme- such as in carrageenin-induced pleurisy (52) as well as ability increase was less in mice with both C1INH in carrageenin-induced rat paw edema (73, 74). As deficiency and bradykinin B2 receptor deficiency (72). shown in Fig. 9, carrageenin-induced pleurisy at 3 h in SD, B/N-Ki, and B/N-Ka rats were compared. B/N-Ka rats showed significantly less exudate volume (Fig. 9A) as well as lower dye exudation rate for 20 min (Fig. 9B) than SD and B/N-Ki rats, but similar leukocyte migra- tion in the pleural cavity (Fig. 9C) as in those two strains. These results suggest a role for endogenous kinin in inflammatory exudation, but not in leukocyte migration. This is a different observation from the data of the B1-receptor knockout mice (12), where migration of neutrophils was affected. Then further examination was performed to obtain proof for kinin liberation during pleurisy. As shown in Fig. 10, immunobloting of H-kininogen in the exudates was conducted. H-kininogen consists of 2 chains, the H- and L-chains, at each side of the bradykinin moiety. After cleavage of the kininogen by plasma kallikrein, the 2 chains remain held together via a S-S bond. When immunoblot analysis was performed with antibody to the light chain after electrophoresis with reduction of the S-S bonds, the light chain and non-cleaved H-kininogen

Fig. 8. Production of permeability factors, PF/Nat and PF/Dil, and TAME esterase and prekalllikrein activator activities in human plasma stored in glass tubes. The graph in panel A illustrates profiles of permeability factors, PF/Nat (permeability factor native) and PF/Dil (permeability factor dilution). Modified from Ref. 65. These factors were produced in guinea pig serum when stored at 2LC for several days in glass tubes at various dilutions, and the potency of the permeability increase was assessed by dye leakage following intradermal injections in guinea pig skin. In panel B, production of esterase activity and prekallikrein activator activity are shown. Data were obtained from an experiment in which human plasma was stored in glass tubes at various dilutions for 44 h at 4LC and then for 1h at 37LC. Esterase activity was assessed by incubation of a fraction of each plasma sample with 3H-TAME (tosyl-arginine methylester). Prekallikrein activator activity was assayed in a incubation mixture of a fraction of each sample combined with partially purified human prekallikrein and then mixed with 3H-TAME (68). Enzyme activities were expressed as arbitrary units based on the hydrolyzed 3H-methanol count. In panel C, prekallikrein-activating activity in various defi- cient plasmas is illustrated. Various plasmas (HANE, normal, pre- kallikrein-deficient, plasminogen-deficient, Factor XII-deficient, and H-kininogen deficient) were stored in glass tubes at various dilutions and assayed in the same way as in panel B. Details are in Ref. 68. Inflammatory Role for the Kinin System 11

Fig. 9. Carrageenin-induced pleurisy in Sprague-Dawley (SD), B/N-Kitasato (Ki), and B/N-Katholiek (Ka) rats. Rats were injected with 2% carrageenin into their pleural cavity and 3 h later, exsanguinated to death. The pleural exudates were then collected and their volumes measured (A). Figures above columns indicate numbers of rats used. Twenty minutes before the sacrifice, pontamine sky blue, 50 mg/kg, was intravenously injected; and dye concentration in the exudates was determined to assess exudated plasma volume for 20 min (exudation rate) (B). Leukocytes in the exudates were counted and classified (C). * indicates statistically significant difference from the corresponding data of Ki at P0.05. Data from Ref. 52.

Fig. 10. Comparison of pleural exudate volumes in the pleurisies induced by kaolin at 3 h, carrageenin at 3 h, phorbol myristate acetate (PMA) at 0.5 h, and zymosan at 5 h in B/N-Kitasato (Ki) and B/N-Katholiek (Ka) rats (A) and immunoblot analysis for H-kininogen in the exudates of B/N-Ki rats (B). Pleural exudates were collected at the indicated times after the stimulator injection, and volumes were measured. Immunoblot analysis was performed with antisera raised in rabbits against the L-chain of rat H-kininogen. * indicates statistically significant difference from the corresponding data of Ki at P0.05. Modified from figures in Ref. 75, and details are in Ref. 75. should be stained. In Fig. 10A, exudate volumes in because only the intact H-kininogen was stained (75). kaolin-induced pleurisy at 3 h and carrageenin-induced These data clearly indicate that endogenous bradykinin pleurisy at 3 h were significantly less in B/N-Ka than was involved in the exudate formation of the pleurisy in B/N-Ki, but no difference was seen in the case of induced by kaolin or carrageenin, but no involvement pleurisy induced by phorbol myristate acetate (PMA) or in the pleurisy induced by PAF, zymosan, or phorbol zymosan. In Fig. 10B, immunoblot analysis of the esters. exudates of B/N-Kitasato after electrophoresis under Next, the effect of a kininase II inhibitor, captopril, reducing conditions clearly indicates that the kinin had on the paw edema induced by 1% carrageenin was been released in the exudate of carrageenin-pleurisy, examined in B/N-Ka and B/N-Ki rats (Fig. 11A). Paw because the L-chain was stained; however, no release swelling of B/N-Ka was significantly less than that of occurred in the zymosan-, PAF-, or PMA-induced one B/N-Ki, and captopril treatment potentiated the swell- 12 A Ueno and S Oh-ishi

Fig. 11. Carrageenin-induced paw edema (A), and pleurisy (exudation and prostanoid production) (B – E) in B/N-Katholiek and B/N-Kitasato rats, and the effect of captopril pretreatment. A: Carrageenin (1%) in saline solution, 0.1 ml, was injected into the sole of the right paw of B/N-Katholiek (Ka) and B/N- Kitasato (Ki) rats; and then, the paw volume was measured at the indicated times after carrageenin injection. Captopril, 10 mg/kg, was intravenously injected 20 min before carrageenin injection. Data are the means with S.E.M. of the indicated numbers of rats. * and ** indicates statistically significant difference from the corresponding data of Ki (open circles) at P0.05 and P0.01, respectively. B – E: Pleurisy was induced by intrapleural injection of 2% carrageenin, 0.1 ml, into B/N-Ka and B/N-Ki rats. Captopril (Ca), 10 mg/kg, was intraperitoneally injected 30 min before the carrageenin injection. The rats were killed by exsanguination at 1 h after carrageenin injection, and pleural exudates were collected. Pleural exudate volume (B) and amounts of prostaglandins (6-keto-PGF1 (C), thromboxane B2 (D), and PGE2 (E)) in the exudates were measured. Data are the means with S.E.M. from the number of rats indicated in the panel B. *significantly different from indicated group at P0.05. Details are in Refs. 73 and 76.

ing in normal rats but had no effect on the kininogen- (78). deficient B/N-Ka rats. Furthermore, captopril enhanced A further example for the enhancement of bradykinin exudate formation in carageenin-induced pleurisy at 1 h by PGI2 is clearly proved by the comparison of paw in B/N-Ki but not in B/N-Ka rats (Fig. 11B) (76). edema induced in IP (prostaglandin I2)-receptor knock- These data proved that endogenous bradykinin is out and wild-type mice (79). As seen in Fig. 12A, IP- involved in the paw swelling induced by carrageenin. receptor knockout mice (IPKO) showed less swelling in The interaction of bradykinin with prostanoids is im- carrageenin- or bradykinin-induced paw edema than did portant patho-physiologically especially in the inflam- wild-type mice (WT), and indomethacin reduced the matory reaction. In carrageenin-induced rat pleurisy, swelling in WT to the level of IPKO, indicating that the co-action of prostanoids, especially PGI2, has been endogenous bradykinin may stimulate PGI2 production revealed. As shown in Fig. 11 (C – E), the amounts of and that these mediators may co-work to enhance the prostanoids measured in the exudates at 1 h of carragee- swelling. A kinin B2 receptor antagonist, FR173657, nin-induced pleurisy increased along with the exudate reduced the swelling of both IPKO and WT to the volume increase by the pretreatment with captopril in same level, clearly indicating that the reduced swelling B/N-Ki but not in B/N-Ka rats (Fig. 11B), indicating in WT is accounted for by the effect of bradykinin itself that endogenous bradykinin may stimulate the produc- plus the enhancing effect of PGI2, while in the IPKO tion of prostanoids. Many previous papers reported the rats, the reduced swelling by the antagonist may account enhancement of pain production (77) and exudation (62) for the almost pure effect of bradykinin. However, a by the co-injection of bradykinin with PGI2 or PGE2 or slight enhancing effect by other prostanoids, if any, the stimulation of prostanoid production by endogenous could not be excluded. In WT, carbacyclin, a synthetic bradykinin in some organs (78). These data all together PGI2 agonist, showed enhancement of the swelling suggest the important inflammatory role of bradykinin induced by bradykinin (Fig. 12C), but in IPKO, no such acting not only by itself but also in conjunction with enhancement was seen (Fig. 12D), and the swelling prostanoids produced by stimulation via bradykinin induced by bradykinin alone was smaller than that of Inflammatory Role for the Kinin System 13

Fig. 12. Involvement of bradykinin and PGI2 in the carrageenin-induced paw edema in mice ((A) and (B)) and enhancement of bradykinin-induced edema by carbacyclin ((C) and (D)). Paw edema was induced by injection of 2% carrageenin (15 l) into the paw of IP-receptor-deficient (KO) and wild-type (WT) mice (open symbols). Indomethacin (closed symbols), 10 mg/kg (A), and bradykinin B2-receptor antagonist FR173657 (closed symbols), 30 mg/kg (B) were intraperitoneally injected 30 min before the carrageenin injection. Bradykinin, 0.3 nmol, was injected with (closed symbols) or without (open symbols) simultaneous carbacyclin, 3 nmol, into the paw of WT (C) and IPKO (D) mice. * and ** express statistically significant difference between treated and control groups at P0.05 and P0.01, respectively. Reproduced from Ref. 79 with permission from Elsevier.

WT. All these results and the previous papers indicate In relation to the role of endogenous bradykinin for that bradykinin exerts its effect for vascular permeability its action on the vasculatures, endotoxin-induced hypo- increase in concert with prostanoids, such as PGI2 and tension in the kininogen-deficient rats was examined PGE2, which are also produced endogenously by stimu- (80). Intravenous injection of endotoxin caused a lation via inflammatory mediators including bradykinin significant hypotension in B/N-Ki, but it was mostly (Fig. 13). suppressed in B/N-Ka rats, and the level was almost

Fig. 13. Interaction of the kallikrein-kinin system with prostanoids. Endogenously produced brady- kinin at an inflammatory site may stimulate the production of prostanoids. Prostanoids produced by cyclooxygenase (COX)-1, COX-2, and terminal prostaglandin syntheses may co-work with brady- kinin to enhance pain sensation and exudate forma- tion in inflammatory tissues. 14 A Ueno and S Oh-ishi similar to that of the B2 antagonist-, Hoe 140, treated types of ion-channels including vanilloid receptor, cap- normal rats, indicating that endotoxin caused the acute saicin-sensitive ion channel, TRY-V1; 2) inflammatory hypotension via the B2 receptor (80). pain, mediated by chemical substances produced in Furthermore, a recently reported notable effect of inflammatory exudates including bradykinin and pros- bradykinin is that bradykinin is protective in some cases taglandins; and 3) neuropathic pain, such as caused by of reperfusion injury of cardiac stenosis or thrombosis spinal cord injury, thalamic stroke, etc. (84). Other (81). Also, a recent interesting report (82) is a study with classifications based on species of transmitters or classes the mixed-gene kininogen deficient rats, which were of anatomical morphology of neurons are also available. produced by back-crossing kininogen-deficient B/N-Ka This review focuses on inflammatory pain propagated and normal B/N rats with genetically susceptible Lewis via polymodal receptors that were previously reviewed rats for 5-generations. In these studies, chronic granulo- by Lim (85) and also finely described by Kumazawa matous enterocolitis was developed in wild-type F5 (86). Lim devised the experimental method of perfusing (F5WT) and kininogen-deficient F5 (F5HKd) rats by bradykinin into the viseral organs, such as the spleen of injection with peptidoglycan-polysaccharide polymers experimental animals, and determined the pain level as into intestinal wall. Total intestinal histologic and liver electrical neuronal activity induced by bradykinin, granuloma scores and other inflammatory changes, which could be suppressed by a local action of aspirin including angiogenesis, in F5HKd were significantly (85). He also applied several other chemical agents, lower than those in F5WT, indicating the importance such as histamine, 5-HT, acetylcholine, substance P, of the kallikrein-kinin system in this model of chronic angiotensin, and vasopressin, into animals and humans enterocolitis (82). These are future targets for drug by various routes and found that bradykinin was the development, in addition to the important involvement most active among them in very small amounts (2 – of bradykinin in the pathogenesis of inflammatory dis- 4 g), which did not appear to cause any sign of injury eases, including some edema, sepsis, and arthritis. (85). He also mentioned that ischemia tended to increase the local acidity, leading to conditions favorable to the 5. Kinin and pain production formation of bradykinin; that local acidosis inhibited kininase, an enzyme that inactivates bradykinin; and that It has been known since the mid 20th-century that bradykinin induced an increase in vascular permeability, bradykinin is one of the potent mediators of inflam- leading to edema, and caused pain, hallmarks of inflam- matory pain (6). Armstrong reported that bradykinin is mation. Furthermore, he suggested that pain perception the most potent nociceptive mediator when assessed by could be mediated through polymodal receptors and the Cantharidin blister-base in human arms (6), and she propagated mainly through C-fibers. Mizumura and and her colleagues suggested that bradykinin could be Kumazawa showed that bradykinin at the dose of produced in vitro in glass tube stored serum and in vivo 0.1 mM induced excitation and facilitation of the pain in tissue subjected to ischemia, injury, or inflammation response via B2 receptors in an experiment using a (83). single polymodal receptor in testis. The duration of the Pain is one of the inflammatory signs, but even so, it response was 15 s for A-delta fibers and 22 s for C- may occur in healthy tissues as an arousing protective fibers. It is an interesting finding that the bradykinin reflex or defensive alert. Strong pain is still one of the response was dependent on the temperature of the most debilitating and significant symptoms needed to be stimulus solution: at 36LC, the response started earlier suppressed in patients suffering from most diseases even and ended faster than at 30LC (86). This fact is well in our modern time, although it has tormented human explained by the recent report of Sugiura et al. (87), beings from the ancient times of history, as already which indicated that bradykinin enhanced the response reported around the era of Avichenna (6). Medicines or of temperature-sensitive ion channel TRPV1 (which is a treatments for pain control, such as morphines and new name for VR1, the vanilloid receptor). aspirin, have been used for a long time, and a multitude Pain assessment in animal inflammatory models has of reviews have been published in the past. Therefore, been developed for the screening of anti-inflammatory only some recent studies are introduced here, and we agents as well as analgesic agents (88). Common models can expect many more in this 21st century as research often used to examine mild inflammatory pain are continues in the pain field in line with the enormous hyperalgesia of carrageenin-induced paw edema of rats development of brain science. and the writhing model of mice; in these models, hyper- Pain receptors may be divided into mainly 3 types algesia could be induced by stimulation of perivascular (84): 1) nociceptive pain activated by heat, acid, and polymodal pain receptors, as shown in Fig. 14. The mechanical force, mainly mediated through various major transmission pathway of inflammatory pain has Inflammatory Role for the Kinin System 15 been documented as the pathway comprising peripheral is endogenously produced and mediates swelling and polymodal receptors that signal the central nervous pain through the B2-receptor in the earlier phase (1 – 4 h) system via sensory C-fibers that enter the dorsal horn of of carrageenin-induced experimental inflammation (89) the spinal cord (86). and also that there is no involvement of B1-receptors, The example shown in Fig. 15, carrageenin-induced because the B2 receptor antagonist suppressed the swell- paw edema of rats, compares the swelling and pain level ing and pain level in B/N-Kitasato to those of B/N- between kininogen-deficient B/N-Katholiek and normal Katholiek rats. The effect of indomethacin suggests the B/N-Kitasato rats when the inflamed paw was subjected involvement of cyclooxygenase products in swelling to laser beam stimulation. B/N-Katholiek rats showed and pain production in this model. significantly reduced swelling in comparison with B/N- Boyce et al. reported that the kinin B2 receptor is Kitasato rats up to 4 h after the carrageenin injection essential to bradykinin-induced acute inflammation such (Fig. 15B), and the level was similar to that of the B2 as carrageenin-induced hyperalgesia, because B2-recep- receptor antagonist (FR173657)-treated normal rats tor null mice showed absence of the nociceptive re- (Fig. 15A). Furthermore, normal B/N-Kitasato rats sponse, whereas the responses to noxious formalin and expressed significantly reduced latent time for with- heat were not different between B2 receptor-deficient drawal following heat stimulation up to 4 h after and normal mice (90). This fact agrees well with the carrageenin stimulation (Fig. 15C); that is, they were result obtained by kininogen-deficient rats (61). susceptible to pain in the inflamed paw, whereas B/N- As for the involvement of B1-receptor in hyperalgesia, Katholiek rats showed almost no reduction in latent there are some controversial reports: Pesquero et al. time, but a similar value as found for the non-inflamed (91) reported that in B1-receptor disrupted mice, hypo- paw. However, no difference in the withdrawal time or algesia was observed by assessment of behavior changes swelling was seen at 24 h in either strain, when the with formalin or capsaicin injection into the paw or paws expressed 30 – 40% swelling in both strains and with the hot plate test. In contrast, Davis et al. (92) no effect was seen with the B2-antagonists nor indo- reported that dorsal root ganglion neurons removed methacin. These results clearly indicate that bradykinin from rats that had been pretreated with Freund’s com- plete adjuvant or carrageenin failed to respond to B1 bradykinin receptor agonists (des-Arg9-bradykinin up to 10- M concentration). In Freund’s adjuvant-induced inflammatory and nociceptive responses, Ferreira et al. (93) reported that B2 receptor knockout mice showed no difference from wild-type mice but that ablation of the B1 receptor gene reduced adjuvant-induced hyper- algesia. While Banik et al. (94) concluded that increased sensitivity of chronically inflamed C-fibers to brady- kinin was B2 receptor-mediated and that B1 receptor may not be important for persistence of inflammation induced by complete Freund’s adjuvant (at 2 – 3 weeks) in rats, they also showed by single fiber recording of mechano-heat sensitive C-fibers in in vitro skin- saphenous nerve preparations that these fibers showed increased sensitivity to bradykinin, which was sup- pressed by a B2 antagonist but not by a B1 antagonist. This discussion shows the need for future extensive Fig. 14. Possible inflammatory pain perceptive pathway and media- research on the induction of the B1 receptor. tors. Inflammatory pain is detected at polymodal pain receptors in The mouse writhing reaction has also been used for local tissues, and the impulses are propagated through the spinal cord evaluation of analgesic activity of agents (95). The by stem to the central nervous system (CNS). Main neurons interven- propionic acid- as well as the acetic acid-induced writh- ing inflammatory pain are known as C-fibers, and some A-delta and A-beta neurons may also be involved. Representative mediators that ing reaction was significantly suppressed by aspirin-like may stimulate polymodal pain receptors are bradykinin, substance P, anti-inflammatory agents and also supressed by brady- prostaglandins, and some cations. Peripheral stimuli produced by kinin B2 receptor antagonists, Hoe 140 or FR173657, these substances, including bradykinin, propagated to the central and by a substance P antagonist; thus endogenous brady- nervous system mainly through C-fibers, because algesic effects of these substances were attenuated in the animals after the degeneration kinin and substance P are mediators of the writhing of C-fibers by neonatal capsaicin-treatment. Details are in Ref. 96. reaction in addition to arachidonic cyclooygenase pro- 16 A Ueno and S Oh-ishi

Fig. 15. Carrageenin-induced paw edema and hyperalgesia in the inflamed paw in B/N-Katholiek and B/N-Kitasato rats. To induce paw edema, 2% carrageenin in saline solution, 0.1 ml, was injected into the sole of the right paw of rats; and then paw volume was measured at the indicated times after the carrageenin injection. Pain level was determined as the difference in withdrawal latency (s) of non-inflamed paw and inflamed paw when the paws received a laser beam stimulation. In some animals, indomethacin (10 mg/kg) or FR173657 (30 mg/kg) was intraperitoneally injected 30 min before carrageenin injection. Data are means with S.E.M. from the number of rats indicated. * and ** express statistically significant difference between treated and control groups at P0.05 and P0.01, respectively. Reproduced from Ref. 89 with permission from Elsevier. ducts (96). The involvement of endogenous bradykinin pH change and acid, and documented as cation channels, was also shown in the kaolin-induced writhing reaction such as TRPs (84). Among those TRPV1, the vanilloid because this reaction was inhibited by co-injection receptor is involved in the acid-induced writhing reac- with SBTI (97). These acid-induced writhing reactions tion (96). Neonatal capsaicin-treatment also reduced the are suppressed significantly by neonatal pretreatment acid-induced writhing reaction, indicating that this reac- with capsaicin, which causes pharmacological C-fiber tion was mainly propagated through C-fibers (96). As degeneration (98). Recent progress in pain receptor shown Fig. 16, the suppressing effect of the bradykinin research includes cloning and identification of tempera- B2-receptor antagonist Hoe 140 was null after capsaicin- ture-sensitive pain receptors, which are also sensitive to treatment, indicating that the bradykinin B2-receptor Inflammatory Role for the Kinin System 17

Fig. 16. Writhing reaction in mice: an inflammatory pain model. Panel A illustrates procedure for neonatal capsaicin treatment. The eye test was performed by counting eye rubbing behavior after dropping 50 l of a 0.01% capsaicin solution into the eye (panel B), and animals that wiped less than 5 times/min were used as effectively capsaicin-treated ones. Effects of various agents on the 1% propionic acid-induced writhing reaction were examined in normal ICR mice (panel C) and neonatal capsaicin-treated mice (panel D). Bradykinin B2-receptor antagonist Hoe 140 at 0.5 mg/kg (Hoe), VR1 receptor antagonist capsazepine at 20 and 40 mg/kg (CPZ), neurokinin receptor (NK1)-antagonist L-732138 at 5 mg/kg (L), and indomethacin at 5 and 10 mg/kg (IND) were subcutaneously injected 30 min before the writhing reaction was induced. Data are means with S.E.M. from the number of rats indicated. ** expresses statistically significant difference between each agent-administered group and the control (C) at P0.05 and P0.01, respectively; and ## expresses statistically significant difference between each agent-administered capsaicin-treated group and the capsaicin-treated control (C) at P0.01. Details are in Ref. 96.

may reside in a C-fiber transmitting pathway. In con- the above tools, along with reference to earlier historical trast, indomethacin further suppressed the capsaicin- information, lead to the following conclusions: brady- treated reduced writhing reaction, suggesting that cyclo- kinin, which is produced by the action of plasma kal- oxygenase products may cause propagation not only of likrein via the initial activation of Factor XII, has an C-fibers but also along other pathways of pain percep- important effect on the formation of inflamatory exu- tion. The important role for prostanoids along with that dates and pain and is the most potent inflammatory of bradykinin has been long known since the discovery mediator. These inflammatory roles of bradykinin were of analgesia by aspirin, and so it is reasonable to expect confirmed to be responsible to 4 inflammatory signs: that prostanoids would enhance bradykinin-induced heat, redness, swelling, and pain; and it was further nociception (99). proved that the bradykinin effect is enhanced by the simultaneously produced prostanoids as well as by the 6. Conclusions low pH of exudates. The latter data were obtained by experiments using prostanoid receptor knockout and The most important research tools available for VR1 knockout mice in addition to the use of the above- evaluation of the roles of the kallikrein-kinin system in mentioned tools. These inflammatory roles can be attri- inflammation are currently bradykinin B1 and B2 recep- buted to mostly B2 receptor-mediated effects, but a tor antagonists, kininogen-deficient B/N-Katholiek rats, clear-cut and commonly acknowledged role for the B1 and bradykinin B1 receptor- or B2 receptor-knockout receptor remains to be defined in the future. The differ- mice. All together the reported data obtained by use of ential roles for B1 and B2 receptors may be elucidated by 18 A Ueno and S Oh-ishi more extensive studies using mouse models of various and their antagponists. Eur J Pharmacol. 1998;348:1–10. diseases. 15 Pixley RA, Colman RW. Factor XII: Hageman factor. Methods Enzymol. 1993;222:51–65. 16 Chung DW, Fujikawa K, McMullen BA, Davie EW. Human Acknowledgments plasma prekallikrein, a zymogen to a serine protease that con- tains four tandem repeats. Biochemistry. 1986;25:2410–2417. The authors are grateful to Emeritus Professors 17 Takagaki Y, Kitamura N, Nakanishi S. 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