Reduction of sodium deoxycholic acid-induced scratching behaviour by bradykinin B2 receptor antagonists

Authors

  • Izumi Hayashi,

    Corresponding author
    1. Department of Pharmacology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228–8555, Japan
      Department of Pharmacology, Kitasato University School of Medicine, 1–15-1 Kitasato, Sagami-hara-shi, Kanagawa 228–8555, Japan. E-mail: hayashii@med.kitasato-u.ac.jp.
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  • Masataka Majima

    1. Department of Pharmacology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228–8555, Japan
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Department of Pharmacology, Kitasato University School of Medicine, 1–15-1 Kitasato, Sagami-hara-shi, Kanagawa 228–8555, Japan. E-mail: hayashii@med.kitasato-u.ac.jp.

Abstract

  • Subcutaneous injection of sodium deoxycholic acid into the anterior of the back of male ddY mice elicited dose-dependent scratching of the injected site with the forepaws and hindpaws.

  • Up to 100 μg of sodium deoxycholic acid induced no significant increase in vascular permeability at the injection site as assessed by a dye leakage method.

  • Bradykinin (BK) B2 receptor antagonists, FR173657 and Hoe140, significantly decreased the frequency of scratching induced by sodium deoxycholic acid.

  • Treatment with aprotinin to inhibit tissue kallikrein reduced the scratching behaviour induced by sodium deoxycholic acid, whereas treatment with soybean trypsin inhibitor to inhibit plasma kallikrein did not.

  • Although injection of kininase II inhibitor, lisinopril together with sodium deoxycholic acid did not alter the scratching behaviour, phosphoramidon, a neutral endopeptidase inhibitor, significantly increased the frequency of scratching.

  • Homogenates of the skin excised from the backs of mice were subjected to gel-filtration column chromatography followed by an assay of kinin release by trypsin from each fraction separated. Less kinin release from the fractions containing kininogen of low molecular weight was observed in the skin injected with sodium deoxycholic acid than in normal skin.

  • The frequency of scratching after the injection of sodium deoxycholic acid in plasma kininogen-deficient Brown Norway Katholiek rats was significantly lower than that in normal rats of the same strain, Brown Norway Kitasato rats.

  • These results indicate that BK released from low-molecular-weight kininogen by tissue kallikrein, but not from high-molecular-weight kininogen by plasma kallikrein, may be involved in the scratching behaviour induced by the injection of sodium deoxycholic acid in the rodent.

British Journal of Pharmacology (1999) 126, 197–204; doi:10.1038/sj.bjp.0702296

Abbreviations:
BK

bradykinin

Introduction

Scratching behaviour is considered to be a mechanism of self-defense when pruritogens invade the body or are produced in it (McMahon & Koltzenburg, 1992). In rodents bioactive substances such as substance P and 5-hydroxytryptamine have been reported to be capable of inducing scratching behaviour when injected into the skin (Kuraishi et al., 1995; Kitagawa et al., 1997). Histamine has also been considered as an important mediator of itching sensations in humans (Hägermark et al., 1978; Fjellner & Hägermark, 1981). Pruritus is an important symptom associated with several disorders, such as atopic eczema (Wahlgren, 1992), cholestasis (Ghent et al., 1977), obstructive jaundice (Herndon & Dallas, 1972), and with dialysis for renal failure (Ståhle-Bäckdahl et al., 1988) and other diseases (Greaves, 1993). In cholestasis or impaired hepatocellular secretion of bile, a marked accumulation in the plasma of bile acids was observed (Ghent et al., 1977; Carey & Williams, 1961). To elucidate a mechanism for the pruritus in cholestasia, bile salts were shown to induce itching sensations in humans (Kirby et al., 1974). On the other hand, antihistamine treatment is widely known to be sometimes ineffective for relief from itching in patients with such symptoms (Krause & Shuster, 1983; Duncan et al., 1984). This suggests a possible involvement of endogenous mediators other than histamine to mediate pruritus (Mettang et al., 1990).

Like histamine and substance P, bradykinin (BK) is known to be one of the most potent endogenous algogenic substances (Dray & Perkins, 1993). Recently, an orally active and long-acting, non-peptide BK B2 receptor antagonist was developed (Asano et al., 1997), that would be useful for evaluating the role of BK in vivo (Rizzi et al., 1997; Majima et al., 1997; Griesbacher & Legat, 1997). Therefore in the present study, we observed the scratching behaviour following s.c. injection of the bile salt, sodium deoxycholic acid in rodents, and examined the effects of BK receptor antagonists and kallikrein inhibitors on scratching behaviour so as to elucidate the possible involvement of BK through activation of the kallikrein-kinin system in pruritus.

Methods

Behavioural experiments

Male ddY mice (Japan SLC, Hamamatsu, Japan, 40–50 g) were used in this study. They were maintained under controlled conditions of temperature (25±1°C), humidity (55±5%), and lighting (lights on only from 06.00  h to 20.00  h). Food and water were consumed freely except during the experiments. Each mouse was put in a clear acrylic cage with a floor area of 170 cm2. The hair at the anterior end of the backs of the mice was cut with an electric shaver before the experiments. Sodium deoxycholic acid (Difco Laboratories, Detroit, MI, U.S.A.) was dissolved in pure water filtrated with MilliQ system (Millipore Corp., Bedford, MA, U.S.A.). Mice were slightly anaesthetized with diethyl ether for the administration of a solution of sodium deoxycholic acid into the hairless area on the back by s.c. injection with a gauge 276G×3/4″ needle. Mice recovered consciousness from the anaesthesia within about 60 s. After the s.c. injection of deoxycholic acid, the mice were replaced in their cages and their behaviours were recorded with an unmanned 8-mm video camera recorder (CCD-TR222, Sony, Tokyo, Japan) for 1 h (Kuraishi et al., 1995). The frequency of scratching around the injected site with fore- and hind-paws was counted by replaying the video recording. Scratching behind the ears, but not the face, was also counted. When the mice scratched continuously for about 1 s without stopping and repeated this more than once, this episode of scratching was counted as one. Inbred female Brown Norway Katholiek rats (plasma kininogen-deficient) and Brown Norway Kitasato rats (normal rats of the same strain), both 3–4 weeks old (40–50 g), kept in the Department of Laboratory Animal Sciences, Kitasato University School of Medicine (Majima et al., 1991), were also used following the same procedures as in the experiment with mice.

All the animal experiments were performed in accordance with Kitasato University School of Medicine guidelines for animal experiments.

Vascular permeability increase

Mice were anaesthetized with 50 mg kg−1 of Nembutal (Abbott Laboratories, North Chicago, IL, U.S.A.) followed by an intravenous injection of pontamine sky blue (100 mg kg−1). BK (Peptide Institute, Osaka, Japan) or sodium deoxycholic acid was injected subcutaneously into the backs of mice. Mice were sacrificed at 30 min after the injection. Skin was removed from the injection site and serum was collected. The excised skin specimens were incubated overnight with 0.5 ml of 1 N potassium hydrochloride at 37°C. Four ml of acetone-0.6 N phosphoric acid (13 : 6) solution was then added and the mixture was centrifuged at 2000×g for 15 min. Concentrations of pontamine sky blue in the supernatant and serum were quantified by measuring the absorbance at 630 nm. The amount of dye leakage in the skin was calculated as the volume of leaked serum (Oh-ishi et al., 1977).

Effects of kininase inhibitors and kallikrein inhibitors on scratching induced by sodium deoxycholic acid

Phosphoramidon (30 μg site−1) or lisinopril (0.3 μg site−1) was dissolved in a solution of sodium deoxycholic acid (0.3 mg ml−1), and 100 μ1 of the mixture was injected locally. The doses of both kininase inhibitors were enough to potentiate an effect of BK, since a vascular permeability increased by s.c. injection of BK (0.1 nmol site−1) into mice was significantly enhanced by co-injections of those kininase inhibitors locally. Soybean trypsin inhibitor (100 μg site−1) was dissolved in a solution of sodium deoxycholic acid (1 mg ml−1). The dose of soybean trypsin inhibitor was also confirmed by a dye leakage method. It inhibited a vascular permeability increase induced by s.c. injection of plasma kallikrein (0.1 unit site−1, Protogen AG., Läutelfingen, Switzerland) in mice.

Effects of kininase inhibitors on degradation of BK by skin homogenate and plasma of mice

Mice were sacrificed and perfused with 50 ml of saline through the abdominal aorta. Skin (5 mm in diameter) was excised and homogenized in 0.5 ml of Tris-HCl (50 mM) buffer (pH 7.5) containing 0.5% of sodium deoxycholic acid. The homogenate was centrifuged at 10,000×g for 15 min and the supernatant was used as skin extract. One μl of the skin extract or 2 μl of plasma was incubated with 100 ng of BK for 10 min at 37°C in 200 μl of Tris-HCl (50 mM) buffer (pH 7.0) containing NaCl (150 mM) in the presence or the absence of lisinopril (1 μg ml−1) or phosphoramidon (100 μg ml−1). One fifth volume of 20% trichloroacetic acid was added to the incubation mixture to stop the reaction. Residual bradykinin in the mixture was measured using a BK enzyme immunoassay kit, Markit-M Bradykinin (Dainippon Pharmaceutical Corp., Osaka, Japan) (Majima et al., 1994a).

Gel-filtration column chromatography

Mice were injected subcutaneously with 100 μl of sodium deoxycholic acid (1 mg ml−1) or 100 μl of saline as vehicle control in the dorsal skin. At 60 min after the injection, the mice were sacrificed and ten pieces of skin, each 5 mm in diameter, were excised from the injection sites. The skin samples were immediately frozen in liquid nitrogen and then pulverized. The pulverized skins were solubilized with 2 ml of Tris-HCl (50 mM) buffer (pH 7.5) containing 1% of sodium deoxycholic acid, 50 μg ml−1 each of aprotinin, soybean trypsin inhibitor, leupeptin, N-ethylmaleimide, phenylsuphonyl fluoride, and disodium ethylenediamine tetraacetic acid. The solutions were sonicated by Sonifier 350 (Branson, U.S.A.), with the output dial set at 6 for 1 min and were centrifuged at 10,000×g for 15 min. The supernatant was applied to a Superose 12 HR 10/30 column (Pharmacia Biotech, Uppsala, Sweden) with a FPLC system. Proteins were eluted with Tris-HCl (20 mM) buffer (pH 7.5) containing NaCl (50 mM) at a flow rate of 0.4 ml min−1. The eluate was collected in 0.4 ml fractions and kinin was released from each fraction. To estimate the kinin released from the gel-chromatography fractions, each fraction was incubated with (100 μg ml−1 each of) trypsin-TPCK (Worthington Biochemical Corp., Free Hold, NJ, U.S.A.), lisinopril (Shionogi & Co. Ltd., Osaka, Japan), phosphoramidon (Peptide Institute, Osaka, Japan), and disodium ethylenediamine tetraacetic acid, at 37°C for 60 min (Hayashi et al., 1984). Following the incubation, one-fifth of the resulting mixture's volume of 20% trichloroacetic acid was added to deproteinize and stop the reaction. The reaction mixture was centrifuged at 2000×g for 10 min and then the amount of kinin released by trypsin was measured using Markit-M Bradykinin.

Materials

FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl] -N-methylaminocarbonylmethyl]acrylamide) and lisinopril were kindly contributed by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan), and Shionogi & Co., Ltd. (Osaka, Japan), respectively. Hoe140 (D-Arg-{Hyp3, Thi5, D-Tic7, Oic8}-BK) and desArg9-[Leu8]-BK were purchased from the Peptide Institute (Minoh, Osaka, Japan).

Statistical analysis

Values are expressed as means±s.e.mean. Student's t-test was used to evaluate the significance of differences. When variances were heterogeneous, statistical analysis was performed by the Aspin-Welch method or Wilcoxon's rank sum test.

Results

Scratching behaviour induced by sodium deoxycholic acid in mice

Following a subcutaneous injection of 100 μg of sodium deoxycholic acid into the anterior part of the backs of ddY mice, scratching behaviour was first observed within a few minutes after the injection (Figure 1). Although varying widely, the frequency of scratching increased over the first 20 min and then gradually decreased for the next 40 min. Intense facial scratching, although not counted, was observed several times preceding scratching around the injection site. Furthermore, the intervals between the scratching episodes were not constant over the period observed. The responses to the scratching-induction effects of different doses of sodium deoxycholic acid were observed over 60 min (Figure 2). Injection of 30 or 100 μg of sodium deoxycholic acid caused an apparent dose-dependent increase in the frequency of scratching. These effects of sodium deoxycholic acid on the scratching were significant (P<0.05 for 30 μg, P<0.01 for 100 μg) when compared with saline alone.

Figure 1.

Time course of scratching after s.c. injection of sodium deoxycholic acid. Sodium deoxycholic acid (100 μg site−1) was injected subcutaneously into the anterior part of the back of male ddY mice under light ether anaesthesia. Each mouse was then placed separately in a cage so that its behaviour could be recorded with an unmanned video camera. Frequency of scratching around the injection site with the fore- and hind-paws was counted for every 5-min period over a 90 min duration after the injection. Each value represents mean±s.e.mean from 9–15  animals.

Figure 2.

Dose-response for the scratch-inducing effect of sodium deoxycholic acid. Ten to 100 μg of sodium deoxycholic acid was injected subcutaneously into the anterior part of the back of male ddY mice. The frequency of scratching around the injection site with the fore- and hind-paws was counted for 60 min after the injection. Each value represents mean±s.e.mean from 9–15 animals. Comparisons were made with the value from mice receiving 100 μl of saline indicated by an open column. *P<0.05; **P<0.01.

Vascular permeability increase due to sodium deoxycholic acid in mouse skin

Doses between 10 and 100 μg of sodium deoxycholic acid did not induce a significant increase in vascular permeability (Figure 3), but 300 μg or more of it did increase vascular permeability (P<0.01). BK (0.3 nmol site−1) also induced dye leakage (P<0.01).

Figure 3.

Vascular permeability increase due to sodium deoxycholic acid in mouse skin. Mice anaesthetized with Nembutal (50 mg kg−1) were administered pontamine sky blue (100 kg mg−1) intravenously followed by s.c. injections of sodium deoxycholic acid (10–1000 μg site−1) intravenously followed by s.c. injections of sodium deoxycholic acid (10–1000 μg site−1), BK (0.3–10 nmol site−1), or saline alone (100 μl) into the dorsal skin. At 30 min after the injection, mice were sacrificed to quantify the amounts of dye that leaked into the skin as described in the text. Each value represents the mean±s.e.mean. Numbers in parenthesis indicate the numbers of experiments. Comparisons were made with the value from mice receiving 100 μl of saline. **P<0.01.

Effects of BK receptor antagonists on the scratching behaviour induced by sodium deoxycholic acid

When the non-peptide antagonist FR173657 was suspended in 5% gum arabic solution and administered orally 1 h before the injection of sodium deoxycholic acid, it inhibited the scratching behaviour significantly (Figure 4, P<0.05). In addition, the B2 receptor antagonist Hoe140, injected subcutaneously with sodium deoxycholic acid, reduced the frequency of scratching by about 30%. In contrast, the B1 receptor antagonist, des-Arg9-[Leu8]-BK did not reduce the frequency.

Figure 4.

Effects of BK receptor antagonists on scratching induced by sodium deoxycholic acid. FR173657 (10–100 mg kg−1) was administered orally 1 h prior to the injection of 100 μg of sodium deoxycholic acid (DC). Hoe140 (30 μg site−1) or des-Arg9-[Leu8]-BK (1 nmol site−1) was injected subcutaneously together with 100 μg of sodium deoxycholic acid. The frequency of scratching around the injection site by the fore- and hind-paws were counted for 60 min after the injection of sodium deoxycholic acid. Each value represents the mean±s.e.mean from 3–15 animals. Numbers in parenthesis are the numbers of mice used. Comparisons were made with the value from mice receiving 100 μg of sodium deoxycholic acid alone, indicated by an open column. *Pgeqslant R: gt-or-equal, slanted0.05.

Effects of kininase inhibitors on scratching induced by sodium deoxycholic acid

Lisinopril, an inhibitor of kininase II, failed to augment the scratching behaviour (Figure 5a). In contrast, phosphoramidon, which inhibits neutral endopeptidase, significantly increased the frequency of scratching induced by sodium deoxycholic acid (P<0.01). Although degradation of BK in plasma was inhibited by lisinopril in vitro, that in skin extract was not (Figure 5b). On the other hand, phosphoramidon inhibited the degradation in skin extract rather than in plasma.

Figure 5.

(a) Effects of kininase inhibitors on scratching induced by sodium deoxycholic acid. Phosphoramidon (30 μg site−1) or lisinopril (0.3 μg site−1) was subcutaneously injected together with 30 μg of sodium deoxycholic acid (DC) into the anterior part of the back of the mice. The frequency of scratching around the injection site by the fore- and hind-paws were counted for 60 min after the injection. Each value represents the mean±s.e.mean from 7–8 animals. Numbers in parenthesis indicate numbers of mice used. Comparisons were made with the value from mice receiving 30 μg of sodium deoxycholic acid alone, indicated by an open column. (b) Effects of kininase inhibitors on degradation of BK by skin extract or plasma of mice. Skin extract or plasma was incubated with BK for 10 min at 37°C in the presence or the absence of lisinopril (1 μg ml−1). Residual bradykinin in the mixture was measured using BK enzyme immunoassay kit. The ordinate indicates residual amounts of BK in the incubation mixture. Each column represents the mean±s.e.mean. **P<0.01.

Effects of kallikrein inhibitors on scratching induced by sodium deoxycholic acid

Simultaneous injection of soybean trypsin inhibitor, to inhibit specifically plasma kallikrein, but not tissue kallikrein, together with 100 μg of sodium deoxycholic acid, did not alter the scratching behaviour (Figure 6). Five minutes before the injection of sodium deoxycholic acid, aprotinin was administered intravenously. Aprotinin decreased the frequency of scratching by approximately 60%. The local administration of aprotinin was abandoned, since it precipitated in 0.1% sodium deoxycholic acid.

Figure 6.

Effects of kallikrein inhibitors on scratching induced by sodium deoxycholic acid. Aprotinin (10 mg kg−1) was administered intravenously 5 min prior to the injection of 100 μg of sodium deoxycholic acid (DC). Soybean trypsin inhibitor (SBTI; 100 μg site−1) was subcutaneously injected together with 100 μg of sodium deoxycholic acid. The frequency of scratching were counted for 60 min after the injection of sodium deoxycholic acid. Each value represents the mean±s.e.mean from 5–15 animals. Numbers in parenthesis indicate the numbers of mice used. Comparisons were made with the value from mice receiving 100 μg of sodium deoxycholic acid alone, indicated by an open column. *P<0.05.

Consumption of kininogen in the mouse skin injected with sodium deoxycholic acid

In a chromatogram of the extract of the skin injected with saline, kinin release was detected in two fractions, as indicated by the bars in Figure 7a, from fraction numbers 28–30, with higher molecular weights and fraction numbers around 39, with lower molecular weights, respectively. Much more kinin was released from the lower molecular weight fractions than from the higher molecular weight fractions. Also, in a chromatogram of the extract of the skin injection with sodium deoxycholic acid, kinin release was again detected in two categories, fractions with higher, and those with lower molecular weights (Figure 7b). Whereas the amounts of kinin released from the higher molecular weight fractions in the skin injected with sodium deoxycholic acid were almost the same as those in the skin injected with saline, the amount of kinin release from lower molecular weight fractions was markedly less for the skin injected with sodium deoxycholic acid than for that injected with saline.

Figure 7.

Superose 12 HR 10/30 column chromatography of the extracts from mouse skin. Mice were injected subcutaneously with 100 μl of saline (a) or 100 μg of sodium deoxycholic acid (b) into the dorsal skin. Ten pieces of skin were excised from each injection site to prepare skin extracts as described in the text. The extracts were applied to a column of Superose 12 HR 10/30 with the FPLC system to separate high-molecular-weight and low-molecular-weight kininogens in the extracts by gel-filtration column chromatography. Proteins were eluted with Tris-HCl (20 mM) buffer (pH 7.5) containing NaCl (50 mM) at a flow rate of 0.4 ml min−1. A sample of 0.4 ml was fractionated. Absorbance of fractions was measured at 280 nm. The measurement of kinin production in each fraction was done by incubation with trypsin followed by BK enzyme immunoassay using Markitt-M Bradykinin. The amount of released kinin in each fraction was calculated as ng BK equivalent.

Comparison of scratching behaviour induced by sodium deoxycholic acid between kininogen-deficient and normal strains of mice

Subcutaneous injection of 100 μg of sodium deoxycholic acid into the anterior of the back of Brown Norway Kitasato rats also resulted in scratching behaviour similar to that in mice (Figure 8). However, the frequency of scratching in the Brown Norway Katholiek rats was significantly less than that in the normal, Brown Norway Kitasato rats (P<0.01). Furthermore, when 10 mg kg−1 of FR173657 was administered orally 1 h before the injection of sodium deoxycholic acid to Brown Norway Kitasato rats, the frequency of scratching declined to that in the Brown Norway Katholiek rats. Pretreatment with FR173657 had no effect on scratching in the latter animals.

Figure 8.

Scratching induced by sodium deoxycholic acid in the kininogen-deficient (B/N-Katholiek) and the normal (B/N-Kitasato) strain of rats. Sodium deoxycholic acid (DC; 100 μg site−1) was injected subcutaneously into the anterior part of the back of female Brown Norway Katholiek (B/N-Ka: kininogens-deficient strain) and Brown Norway Kitasato (B/N-Ki; the normal strain) rats. The frequency of scratching around the injection site by the fore- and hind-paws was counted for 120 min after the injection. Each value represents the mean±s.e.mean. The numbers in parenthesis indicate the numbers of rats used. The last two columns indicate the result following administered of FR173657 (10 mg kg−1) orally 1 h before the injection of sodium deoxycholic acid. Comparisons were made with the value from Brown Norway Kitasato rats receiving 100 μg of sodium deoxycholic acid shown in the left-hand. *P<0.05; **P<0.01.

Discussion

The retention of bile salts is thought to be the cause of pruritus in cholestasis and obstructive jaundice (Herndon & Dallas, 1972). Although the intravenous or oral administration of exogenous cholate failed to produce any itching (Osborn et al., 1959; Carey, 1958), application of bile salts to keratin-stripped skin (Varadi, 1974) and to cantharidin-induced blister bases on the forearm (Kirby et al., 1974) induced pruritus in human subjects. Firstly, we tested whether the administration of bile salts has the ability to induce itching in experimental animals as well as in man. Rothman (1941) defined an itch as an unpleasant cutaneous sensation which provokes the desire to scratch in man. The present results indicate that bile salts can elicit scratching in mice as well as in man. The concentrations of sodium deoxycholic acid which induced scratching corre-spond well with the concentrations at which purified bile salts were effective in producing pruritus in humans (Kirby et al., 1974). In the present model, s.c. injection of up to 100 μg of sodium deoxycholic acid did not cause a significant increase in vascular permeability though 300 μg and greater did. Up to 100 μg of sodium deoxycholic acid had almost no effect on plasma extravasation, suggesting that the contribution of plasma proteins to the scratching behaviour is very small.

Although bile salts liberate small amounts of histamine when perfused into animal skin (Shachter, 1952), and blood histamine levels increase in experimental obstructive jaundice (Anrep & Barsoum, 1953), the poor therapeutic response to antihistamine suggests that histamine is not the major mediator of the pruritus (Krause & Shuster, 1983; Duncan et al., 1984). These observations suggest that endogenous chemical mediators other than histamine might be involved in the pruritus associated with cholestasis (Jones & Bergasa, 1992; Khandelwal & Malet, 1994). Scratching behaviour is reported to be produced by s.c. injection of pruritogenic agents, such as capsaicin and formaldehyde, in mice (Kuraishi et al., 1995). We have presented three pieces of evidence to suggest a partial involvement of kinin in the scratching behaviour induced by sodium deoxycholic acid. Firstly, two BK B2 receptor antagonists markedly reduced the frequency of the scratching. Since both Hoe140 and FR173657 have been clearly characterized as specifically inhibiting the action of BK by blockade of the B2 receptor (Wirth et al., 1991; Asano et al., 1997), the reduction of the scratching behaviour by these antagonists indicates the involvement of endogenous BK and the B2 receptor in this scratching model. Secondly, treatment with phosphoramidon to inhibit neutral endopeptidase, which degrades kinin (Ura et al., 1987; Kuribayashi et al., 1993), significantly augmented the scratching behaviour, suggesting a potentiation of the action of kinin released due to the injection of bile salt. Thirdly, when the production of kinin was suppressed by tissue kallikrein inhibitor, aprotinin (Trautschold et al., 1967), an apparent reduction of the scratching behaviour was observed, indicating an endogenous production of kinin in this model. Furthermore, to support the participation of kinin in the scratching behaviour in another rodent, we used two strains of rats. A clearly milder scratching response was observed in the kininogen-deficient Brown Norway Katholiek rats than in normal Brown Norway Kitasato rats. In the former rats, neither high-molecular-weight kininogen nor low-molecular-weight kininogen are secreted from the hepatocytes where kininogens are normally biosynthesized, so that almost no kinin release occurs (Hayashi et al., 1985, 1989, 1993; Hayashi & Oh-Ishi, 1993). In addition, the frequency of the scratching in the Brown Norway Kitasato rats was decreased by treatment with FR173657 to almost the same level as was seen in the Brown Norway Katholiek rats. These results indicate the involvement of kinin in the scratching behaviour of normal rats.

It is well known that there are two cascade reactions, i.e., the plasma kallikrein-kinin system and the tissue kallikrein-kinin system (Movat, 1979; Fiedler, 1979). We have already reported that BK released from high-molecular-weight kininogen via the activation of the plasma kallikrein-kinin system plays an important role in increasing vascular permeability during some acute inflammatory reactions (Oh-Ishi et al., 1986, 1987; Fujie et al., 1993; Majima et al., 1997). On the other hand, kinin released from low-molecular-weight kininogens via activation of the tissue kallikrein-kinin system has the crucial function of mediating the excretion of accumulated sodium ions in the kidneys to maintain the balance of electrolytes in the body (Majima et al., 1991, 1993, 1994b). Thus, these kinin-generating systems are controlled separately and function independently.

Which kinin system plays a role in the scratching behaviour induced by sodium deoxycholic acid was also clarified in the present study. Four results indicate the tissue kallikrein-kinin system is responsible for producing the kinin in the scratching behaviour. Firstly, as mentioned above, up to 100 μg of sodium deoxycholic acid did not induce a significant increase in vascular permeability when subcutaneously injected. Because of less plasma leakage into the injected site, activation of the plasma kallikrein-kinin system at the site is unlikely. Secondly, lisinopril, which inhibits kininase II, did not affect the frequency of the scratching. This result would also suggest to less plasma leakage at the injection site, since kininase II is a dominant kinin-degrading endopeptidase in the circulation rather than in the tissue. In contrast, neutral endopeptidase inhibitor, phosphoramidon significantly increased the scratching behaviour. This endopeptidase has been reported to be a kinin-degrading enzyme localized in tissues such as the kidney of the rat (Ura et al., 1987). Thirdly, the frequency of scratching was reduced when the production of kinin was suppressed following inhibition of tissue kallikrein activity by aprotinin, whereas soybean trypsin inhibitor, which inhibits plasma kallikrein activity (Wunderer et al., 1972), did not have any effect. Fourthly, gel-filtration experiments showed less kinin release from the fractions eluted as a lower molecular weight of skin extract after sodium deoxycholic acid injection. This suggests the consumption of low-molecular-weight kininogen rather than high-molecular-weight kininogen as this eluted in the fractions with a higher molecular weight. If all of these resuls are taken together, kinin released from low-molecular-weight kininogen by tissue kallikrein could be involved in part of the scratching behaviour induced by sodium deoxycholic acid.

Bradykinin has often been characterized as a mediator of pain and hyperalgesia through its effects on various neural tissues and sensory neurons (Keele & Armstrong, 1964; Steranka et al., 1988; Dray & Perkins, 1993). On the other hand, intradermal injection of BK or kallikrein on the upper arms was also reported to cause itching in human subjects (Hägermark, 1974). Whereas one site of action of BK in producing pain has been considered to be the C-fibre nociceptors (Steranka et al., 1988; Lang et al., 1990), the excitation of afferent C-fibres in the skin by the stimulation of spicules of cowhage, which causes itching, has been reported (Tuckett & Wei, 1987a,1987b). Therefore, it is possible that BK mediates not only pain production but also itch production. The details of the mechanisms of tissue kallikrein-kinin system activation by bile salts and the precise mechanisms that mediate the scratching behaviour remain to be elucidated. However, the observations in the present study also raise the possibility of a therapeutic use of a BK B2 receptor antagonist as an antipruritic drug.

Acknowledgments

The authors are indebted to Dr N. Inamura (Fujisawa Pharmaceutical Co., Osaka, Japan) and Dr N. Sunahara (Dainippon Pharmaceutical Corp., Osaka, Japan) for their respective generous gifts of FR173657 and MARKIT-M Bradykinin. We also thank Mr C.W.P. Reynolds for correcting the English of this manuscript. Part of this work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture #09772057 (to I.H.) and #07672472 (to M.M.). Part of this study was also supported by a grant from Fujisawa Pharmaceutical Co. to M.M.

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