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Contribution of bradykinin to heat-induced substance P release in the hind instep of rats
Life Sciences.,Vol. 56, No. 20, pp. 1679-1688,199S CopyAght Q 1995 FWvier Science Ltd PI!inti in the USA. All rights rcscrvcd CTu24-3205/95$950 + .oo
0024-3205(95)00136-O
CONTRIBUTION OF BRADYKININ TO HEAT-INDUCED SUBSTANCE RELEASE IN THE HIND INSTEP OF RATS
Norifumi
Yonehara*, Kihachi Saito, Sachiko Makoto Katori and Reizo Inoki
P
Oh-ishi
Department of Pharmacology (N.Y., K.S., R.I.), Faculty of dentistry, Osaka University, Suita 565, Osaka, Japan Department of Pharmacology (S.O.), School of Pharmaceutical (M.K.) School of Sciences, and Department of Pharmacology Medicine, Kitasato University, Sagamihara, Kanagawa 228, Japan (Received in final form February 27, 1995)
Summary
contribution of the investigate the possible To (BK) system to heat-induced substance P bradykinin primary (SP) release from the peripheral endings of molecular weight afferent nerves, we used the high low molecular weight and (LMW) kininogen(HMW) deficient rat strain (Brown Norway-Katholiek, B/N-Ka) (Brown Norway-Kitasato, normal rat strain and the We found that immersion of the paw of B/NB/N-Ki). 20 min for led to rats in water of 47 OC Ki significant SP and Evans blue increases of BK, extravasation in the S.C. perfusate, and that similar resulted in significantly lower levels in treatment Local application of BK (10-d M) to the B/N-Ka rats. infusion of BK and intra-arterial S.C. perfusate mol/kg) increased Evans blue extravasation and 10-S SP release evoked by heat stimulation, respectively, in B/N-Ka rats to similar levels to those in B/N-Ki treatment. rats after heat-stimulation without BK These results indicate that BK released into the noxious stimulation is extravascular space by involved in SP release from the peripheral endings of capsaicin-sensitive primary sensory neurons. Key Words: substance P, bradykinin, kininogen-deficient
rat strain
Bradykinin (BK), a g-amino acid peptide, is one of the most potent pain-producing substances that appear in injured tissue by enzymatic cleavage of a large plasma protein (1). In fact, high * To whom
all correspondence
should
be addressed
1680
SubstanceP Release and Bradykinin
Vol.54,No.20,1!X'S
levels of BK appear in damaged tissue, especially when there is a BK has direct effects on visible inflammatory exudate (2,3). where it acts via BK receptors, the BK vascular permeability(4), receptors are localized on sensory nerve terminals, as shown by autoradio-graphy of [3H] -BK and BK causes excitation of nociceptors (5). Some of the information about the stimulus transduced by activation of that is nociceptors is converted to a form accesible to the brain and is transferred to the dorsal horn of the spinal cord. In addition, some of the information is transmitted to the periphery through axon reflex phenomena: that is, primary afferent fibers serve efferent as well as afferent functions. With to regard the former function, recent pathophysiological studies in humans and laboratory animals have indicated that primary afferent fibers containing neuropeptides such as tachykinin and calcitonin gene-related peptide (CGRP) may contribute to the pathogeneses of thermal injury, polyarthritis, respiratory-tract irritation, etc. (6-9). In this connection, in the skin of the rat hind paw, we observed that noxious heat stimulation led to the releases of substance P (SP) and CGRP from the small-diameter afferent fibers and that these neuropeptides released in the periphery are closely related to edema formation in the thermal injury reaction (10,ll). These findings suggest that nociceptor activity is not merely a passive signal indicating that tissue damage has occurred, but may also play an active role in body defense by participating in the inflammatory process; that is, afferent impulses from nociceptors generated by a noxious stimulus cause release of neuropeptides, which finally control the inflammatory response. aim of this study was to demonstrate more clearly the The peripheral involvement of kinins in the SP released from the noxious heat afferent fibers following endings of primary weight (HMW) stimulation. For this, we used the high molecular strain rat low molecular weight (LMW) kininogen-deficient and normal rat strain Norway-Katholiek (B/N-Ka)) and the (Brown (Brown Norway-Kitasato (B/N-Ki)), and determined the releases of extravasation and BK into the S.C. perfusate and Evans blue SP evoked by noxious heat stimulation.
Materials
and Methods
rats were obtained and B/N-Ki B/N-Ka Kitasato School of Medicine, laboratory of strains were bred and maintained in the animal faculty, Osaka University.
animal from the Both University. laboratory of our
with urethane (780 B/N-Ka and B/N-Ki rats were anesthetized introduced A double coaxial polyethylene tube was i.p.). the containing saline into the S.C. space of the instep, and and the bestatin (3 mg/lOO inhibitor aminopeptidase ml)
mg/kg
Vol.56,No.20,1995
SubstancePReleaseandBradykinin
1681
dipeptidyl inhibitor, captopril carboxypeptidase (0.1 mM) was perfused through this tube with a peristaltic pump at a rate of 0.1 ml/min. The perfusate was collected through the outer tube in lo-min fractions in test tubes in an ice bath using a fraction collector. The samples were lyophilized and then radioimmunoassayed for BK and SP using the corresponding antisera. Details of the radioimmunoassay procedure of SP were as described previously (12). For the assay of BK, the perfusate was collected in test tubes containing two volumes of ethanol in an ice bath. The mixture was centrifuged at 3400 g for 15 min and 2.5 ml of supernatant was lyophilized. After lyophilization, each lo-min fraction was redissolved in 200 1-r1 of phosphate buffered (10 m&l, pH 7.4) containing 0.1 % gelatin, and saline then 10000 100 111 of [3H]BK, adjusted to approximately cpm per tube, and 100 1~1 of anti-BK rabbit serum (dilution 1 in 15000) were successively added. The polyethylene test tubes containing 4 OC for 12-18 h. sample solution were incubated at For separation of bound and free radiolabeled peptides, 250 1~1 of 1 % of dextran-coated charcoal was added to each solution tube and incubation continued at 4'C for 30 min. Thereafter, was the 15000 g for 20 min at 4 OC. tubes were centrifuged at The 650 ~1 of the supernatant was measured in a radioactivity of liquid scintillation counter. The antiserum of BK showed crossreactivities IleLys-BK, of 68 %, 60 % and less than 1 90with Ser-BK and SP, respectively. For measurement of vascular permeability, the S-C. space of perfused as described above. Evans blue the instep was (50 urethane mg/kg) was injected i.v. into animals anesthetized with side (780 mg/kg i.p.), and 20 min later the paw on the perfused The amount of immersed for 20 min in a water-bath at 47'C. was released into the perfusate (collected in lo-min fractions) dye a wavelengh 620 nm with was measured at a spectrophotometer Shimadzu Scientific Instruments, Kyoto, Japan). (model W-240, are shown as mean I_ SEM for the number of animals Results indicated. statistical analysis Student's t-test for For P values of unpaired values was less than 0.05 were used. considered to be significant. Materials: The following drugs were used: BK and SP from Peptide Institute (Japan), BK antiserum (a gift from Nippon Zoki Pharmaceutical co. (Hyogo, Japan)), and [125I][Tyr8]SP and [2,3prolyl-3,43H(N)]-BK from DuPont/New England Nuclear Research Products (USA).
Results Effect of heat stimulation and B/N-Ki rats
an
on the release
of Evans
blue
in B/N-Ka
As can be seen in Fig. 1, noxious heat stimulation caused increase in Evans blue release in the both B/N-Ki rats and
1682
Vol. 56, No. 20, 1995
Substance P Release and Bradykinin
B/N-Ka
rats, but the stimulus-evoked Evans blue release, was than in the significantly less in the B/N-Ka strain BIN-Ki strain: each amount of Evans blue released in the two 10 min fractions during stimulation in B/N-Ki (n=lO) and B/N-Ka rats (n=18) were 2.81 ?r 0.1 i_rg/kg (first lo-min samples) and 3.92 + 0.13 pg/kg (second lo-min samples), and 0.92 + O.ll.rg/kg (first lo-min lo-min samples) and 2.25 + 0.12 pg/kg (second samples), respectively. Local application of BK (lo-4 M) to the S.C. perfusate of the B/N-Ka strain significantly enhanced the stimulus-evoked Evans blue release, the maximum increase being almost the same as that in the B/N-Ki strain (4.12 f 0.1 I-lglkg, n=15).
o--O BIN-Ki O--O BIN-Ka
I
H
E/N-Ka
(BK 10X4
5 t
OO-
1
2
3 -(hr)
heating EK (10-M)
Fig.
1
Effect of heat stimulation on the release of Evans instep of blue into the S.C. perfusate in the hind and effect of local B/N-Ki and B/N-Ka rats, B/N-Ka BK to the S.C. perfusate of application of rats. Values are means of at least 10 experiments. range of The deviation of each value is within the value each symbol. * P co.05 when compared with the obtained in the B/N-Ka rats at each time. Effect rats
of heat
stimulation
on the releases
of SP and BK in B/N-Ki
As shown in Fig. 2(A), BK release did not increase in the markedly for lo-20 min but increased first lo-min samples, (second lo-min samples) after onset of immersion of the hind paw in hot water at 47'C. On the other hand, heat stimulation for 20 time: that is, a significant increase of SP with min led to marked release of SP was detected immediately after the start of
Vol. 56, No. 20, 1995
noxious rapidly
1683
Substance P Release and Bradykinin
heat stimulation and this stimulus-evoked SP release decreased after the end of stimulation (Fig. 2(B) ).
was
(A)
1oll~ L
.9
JO 60
120
160
(mid
120
180
(mid
heating
(B) 2000 0
z
2
m, 1500-
a :
5
lOOO-
; 4 n ap
500-
lo;-
Q 60
1 heating
Fig.
2
Effect of noxious heat stimulation on the releases of BK (A) and SP (B) into the S.C. perfusate .in the hind instep of BIN-Ki rats, and change in the subcutaneous noxious For temperature (upper line in (A)). stimulation, a rat paw was immersed in a water-bath change in For calculating the at 47OC for 20 min. the four release, the mean value for peptide the fractions collected before heating was taken as control value (100%). The peptide contents in each successive fraction are expressed as percentages of Each value represents the mean f this control value. SEM of 16 experiments. Release of BK induced Ka rats As
shown
by heating
in Figs.
the hind paw of B/N-Ki
2 and 3, immersion
of the hind
and
paw
B/N-
of
1684
Substance P Release and Bradyldnin
Vol. 56, No. 20, 1995
B/N-Ki rats in hot water caused a significant increase of BK in while similar treatment of B/N-Ka the perfusate, rats caused significantly less increase of BK. The average amounts of BK released in the two fractions obtained in the 20-min collection periods before and during noxious heat stimulation were, respectively, 0.124 + 0.015 pmol/lO min and 0.430 + 0.06 pmol/lO min in B/N-Ki rats, and 0.008 f 0.002 (n=15) pmol/lO min and 0.012 + 0.002 pmol/lO min (n=16) in B/N-Ka rats. Release of SP induced Ka rats
by heating
the hind paw of B/N-Ki
and
B/N-
The SP levels in the resting state; that is, the average amount of SP released in the fractions in the two 20-min collection noxious period before heat stimulation were 0.017 f 0.004 pmol/lO min (n=lO) in B/N-Ki rats and 0.019 f 0.003 fmol/lO min (n=lO) in B/N-Ka rats. As shown in Fig. 4, noxious heat stimulation produced a marked increase of SP release in both strains, but the SP level in the B/N-Ka strain (0.069 + 0.014 pmol/lO min, n=lO) was significantly lower than that in the B/NKi satrain (0.121 + 0.014 pmol/lO min, n=lO). To determine whether release of SP into the subcutaneous perfusate evoked by heat stimulation was modulated by BK, we infused BK into the foot on the side of perfusion through an arterial cannula inserted into the contralateral iliac common artery of B/N-Ka rats. At the concentration employed BK enhanced the heat-evoked SP release to almost (10 umol/kg), the same level as that in B/N-Ki rats after heat stimulation without BK treatment.
Discussion plasma Recent study on rat kininogen demonstrated that rat and kallikrein releases BK from high molecular weight kininogen, from high and low kallikrein releases also BK rat urinary there is no In addition, weight kininogens molecular (13). Taking sequence in rat kininogens by cDNA study (14). kallidin showed these findings into account, although the antiserum of BK Ile-Ser-BK, of 68 % and 60 % with Lys-BK and cross-reactivities that a large be reasonable to consider respectively, it may portion of kinins detected in the perfusate is BK. As shown in Fig. 2A, an increase in BK release was observed in the second lo-min samples, i.e. 10 - 20 min after the start of first immersion of the hind paw in water at 47"C, but not in the observation This result was consistent with the lo-min samples. that the showed Sato and Yamaguchi Starr and West (15). of release enzyme and kininase increase of kininogen, kinin-forming heat onset of behind the also lagged perfusate into the On the other hand, a marked increase in SP stimulation (16). heat start of after the immediately detected release was stimulation and this stimulus-evoked SP release rapidly decreased the difference between The stimulation. the end of after
Vol.56,No.U), 199.5
Fig.
Substance P Release and Bradykinin
1685
3 0.6L
Effects of noxious heat stimulation on the releases of BK into the S.C. perfusate in the hind instep of B/N-Ki and B/N-Ka rats. Each value represents the mean + SEM for BK release in the two fractions obtained in 20-min collection periods before, during and after heat stimulation (stim.), in at least 15 experiments.
0
E/N-Ki
Q
B/N-Ka
PCO.05
I
I
OS”
2 0.4,a .c .G 5 0.3Ti m 2 0.2-
0.1-
BIN-Ki
before
during
after
stim.
stim.
stim.
B/N-Ka BIN-Ka (BK lO_‘mol/kg
before
stim.
during stim.
Fig.
i.a.)
after stim.
4
Effect of noxious heat stimulation on the release of SP into the S.C. perfusate in the hind instep of B/NKi and B/N-Ka rats, and influence of BK infused into artery of B/N-Ka rats on its the common iliac release. Each value represents the mean f SEM for SP obtained in release in the two fractions 20-min collection periods before, during and after heat stimulation (stim.), in 10 experiments. release of patterns of BK and SP may be attributed to the difference between the responses to heat stimulation of these
1686
Substance P Release and Bradykink
Vol. 56,No. 2Ql995
It is known that BK is released peptide-releasing systems. from called kininogens - by a precursor in plasma - a2-globulins the action of proteolytic enzymes with the specificity of trypsin we have demonstrated that SP into the released (171, whereas subcutaneous perfusate following heat stimulation originates mainly from the peripheral endings of capsaicin-sensitive, smalldiameter afferent fibers (10). the B/N strain of rats were Two types of used in the present study, the mutant rat strain named Brown Norway Katholiek (B/N-Ka) and the normal strain, Brown Norway Kitasato (B/NKi) (18,19). The former is deficient in high and low molecular weight kininogens in the plasma, and the level of prekallikrein is only about 30 % of the latter (20). In the present study, the amounts of BK released into the perfusate both before and during noxious heat stimulation were much lower in B/N-Ka rats than in B/N-Ki rats. In addition, B/N-Ka rats showed significantly less heat-evoked extravasation than B/N-Ki rats, but the application of BK to the S.C. perfusate of B/N-Ka rats increased heat-evoked extravasation to almost the level observed in B/N-Ki rats. These results indicate that BK detected in the extravascular space may be largely produced through activation of the plasma kallikreinkinin system, and this plasma kinin system has an important role in the thermal injury reaction. Concerning the functional interaction between BK and SP released by noxious heat stimulation, the amount of the heatwas significantly lower in the evoked SP release kininogenstrain B/N-Ka, than in B/N-Ki rats. deficient Furthermore, as shown in Fig. 4, infusion of BK into the common iliac artery of rats, as in the extravasation experiment (Fig. l), B/N-Ka enhanced the level of the heat-evoked SP release to nearly the that in normal rats. findings suggest the level of These contribution of the plasma kinin system to SP release induced by been In this connection, it is noteworthy that BK has heating. also shown to act not only as a powerful pain-inducing agent but topical In fact, as an important mediator in inflammation (21). administration of BK increases blood flow and vascular permeabilperipheral tissues including the skin (4) and ity in most by and at least part of the response is mediated airways (221, For small-diameter afferent fibers capsaicin-sensitive, (23). evoked by BK instilled into guineaexample, broncho-constriction can be abolished by capsaicin pre-treatment (24). airways pig Ul\ caused % of the extravasation almost 60 Furthermore, topically into the nose of rats was inhibited b;' CPinstilled 96345, a selective inhibitor of NKl receptors (25). BK receptors have been localized to nociceptive pathways (peripheral terminals small small-diameter afferent fibers and of capsaicin-sensitive, cells in dorsal root ganglia) by autoradiography of L3~l-~~ (5). saphenous nerve the release of SP from evoked Moreover, BK that neuromas of the rat (26). We also have already demonstrated heating induced by of SP and the edema formation release the pretreatment simultaneous significantly by the inhibited were bromelain (which causes a specific depletion of HMW with stem kininogen) and emorfazone (a nonacidic antiinflammatory drug that
Vol.56,No.20,1995
Substance P Release andBradylcinin
1687
inhibits the release of BK into extravascular space (16)), and by the intra-arterial infusion of Des-Arg9-[Leu8]BK (BK antagonist) These observations and our present results strongly (10,271. the hypothesis that BK released into extravascular support the noxious heat stimulation causes excitation of BK space by receptors in sensory nerve terminals that, in turn, activates capsaicin-sensitive, small-diameter afferent fibers to release SP from peripheral endings through a mechanism such as an axonreflex arrangement. In the present experiment, noxious heat stimulation of B/NKa rats induced a very low level of BK release, but a significant increase in SP release. Furthermore, in the first lo-min sample immediately from normal rats, a large release of SP was detected after the start of the experiment without BK release, suggesting substances other than BK may be involved in SP that chemical especially in its initial stage of release, evoked by release, the peripheral endings of noxious heat stimulation from capsaicin-sensitive primary afferent fibers.
Acknowledgements
their
We would generous
like to thank Drs. Yasuo Imai and Sou Takiuchi assistance with this project.
for
References 1. 2. 3. 4. 5.
6.
7. 8. 9. 10. 11. 12. 13. 14. 15.
G.P. LEWIS, Handb. exp. Pharmacol. 25 516-530 (1970) STARR and G.B. WEST, Brit. JF Pharmacol. 178-187 M.S. -31 (1967) D. ARMSTRONG, Handb. exp. Pharmacol. 25 434-481 (1970) Z. KHALIL and R.D. HELME, Brain Res. 589 102-108 (1992) L.R. STERANKA, D.C. MANNING, C.J. DEHAAS, J.W. FERKANY, S.A. CONNOR, R.J. VAVREK, J.M. STEWART and S.H. BOROSKY, J.R. SNYDER, Proc. Natl. Acad. Sci. USA, 85 3245-3249 (1988) F. LEMBECK, Substance P: metabolism and biological actions, C.C. Jordam and P. Oehme (Eds), 137-151, Taylor and Francis, London (1985) X.-Y. HUA, Acta Physiol. Stand. Suppl. 551 1-45 (1986) C.A. MAGGI and A. MELI, Gen. Pharmacol. 19 l-43 (1988) K.A. SHARKEY, Ann. N. Y. Acad. Sci. 664 425-442 (1992) N. YONEHARA, T. SHIBUTANI and R. INOKI, J. Pharmacol. Exp. Ther. 242 1071-1076 (1988) N. YONEHARA, Y. IMAI and R. INOKI, Japan. J. Pharmacol. 56 381-384 (1991) N. YONEHARA, T. SHIBUTANI, H.-Y. TSAI and R. INOKI, Eur. J. Pharmacol. 129 209-216 (1986) H. KATO, K. ENJYOJI, T. MIYATA, I. HAYASHI and S. OH-ISHI, Biochem. Biophys. Res. Commun. 127 289-295 (1985) S. FURUYO-KATO, A. MATSUMOTO, N. KITAMURA and S. NAKANISHI, J. Biol. Chem. 260 12054-12059 (1985) M.S. STARR and G.B. WEST, Brit. J. Pharmacol. 31 178-187 (1967)
1688
16.
17. 18. 19. 20. 21. 22. 23. 24. 25.
26. 27.
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Vol. 56, No. 20, 1995
Res. -32 379M. SAT0 and A. YAMAGUCHI, Arzneim-Forsch./Drug 382 (1982) ROSENFELD, Am. J. ROCHA E SILVA, W.T. BERALDO and G. M. Physiol. 156 261-273 (1949) S. OH-ISHI, I. HAYASHI, K. SATOH and T. NAKANO, Thrombosis Res. 33 371-377 (1984) I. HAZSHI, S. OH-ISHI, H. KATO, K. ENJYOGI, S. IWANAGA and T. NAKANO, Thrombosis Res. 39 313-321 (1985) S. OH-ISHI, K. SATOH, I. HAYASHI, K. YAMAZAKI and T. NAKANO, Thrombosis Res. 28 143-147 (1982) D. REGOLI and J.>ARABE, Pharmacol. Rev. 32 1-46 (1980) J.O. LOTVALL, K. TOKUYAMA, P.J. BARNES and K.F. CHUNG, Eur. J. Pharmacol. 200 211-217 (1991) JANCSG, E. KItiLY and A. JANCSG-GABOR, Int. J. Tissue G. React. 2 57-66 (1980) M. ICHINOSE, M.G. BELVISI and P.J. BARNES, J. Pharmacol. Exp. Ther. 253 594-599 (1990) GEPPETTI, J. BAKER, G. PETERSSON, G. BERTRAND, P. C. 2456-2461 J.A. NADEL, J. Appl. Physiol. PIEDIMONTE and -74 (1993) Lett. 108-113 WHITE and M. ZIMMERMANN, Neurosci. D.M. -92 (1988) N. YONEHARA, Y. IMAI, T. SHIBUTANI and R. INOKI, KININS V, Fujii 529-534, PART B, K. Abe, H. Moriya and S. (Eds), Plenum Publishing, New York (1989)
0024-3205(95)00136-O
CONTRIBUTION OF BRADYKININ TO HEAT-INDUCED SUBSTANCE RELEASE IN THE HIND INSTEP OF RATS
Norifumi
Yonehara*, Kihachi Saito, Sachiko Makoto Katori and Reizo Inoki
P
Oh-ishi
Department of Pharmacology (N.Y., K.S., R.I.), Faculty of dentistry, Osaka University, Suita 565, Osaka, Japan Department of Pharmacology (S.O.), School of Pharmaceutical (M.K.) School of Sciences, and Department of Pharmacology Medicine, Kitasato University, Sagamihara, Kanagawa 228, Japan (Received in final form February 27, 1995)
Summary
contribution of the investigate the possible To (BK) system to heat-induced substance P bradykinin primary (SP) release from the peripheral endings of molecular weight afferent nerves, we used the high low molecular weight and (LMW) kininogen(HMW) deficient rat strain (Brown Norway-Katholiek, B/N-Ka) (Brown Norway-Kitasato, normal rat strain and the We found that immersion of the paw of B/NB/N-Ki). 20 min for led to rats in water of 47 OC Ki significant SP and Evans blue increases of BK, extravasation in the S.C. perfusate, and that similar resulted in significantly lower levels in treatment Local application of BK (10-d M) to the B/N-Ka rats. infusion of BK and intra-arterial S.C. perfusate mol/kg) increased Evans blue extravasation and 10-S SP release evoked by heat stimulation, respectively, in B/N-Ka rats to similar levels to those in B/N-Ki treatment. rats after heat-stimulation without BK These results indicate that BK released into the noxious stimulation is extravascular space by involved in SP release from the peripheral endings of capsaicin-sensitive primary sensory neurons. Key Words: substance P, bradykinin, kininogen-deficient
rat strain
Bradykinin (BK), a g-amino acid peptide, is one of the most potent pain-producing substances that appear in injured tissue by enzymatic cleavage of a large plasma protein (1). In fact, high * To whom
all correspondence
should
be addressed
1680
SubstanceP Release and Bradykinin
Vol.54,No.20,1!X'S
levels of BK appear in damaged tissue, especially when there is a BK has direct effects on visible inflammatory exudate (2,3). where it acts via BK receptors, the BK vascular permeability(4), receptors are localized on sensory nerve terminals, as shown by autoradio-graphy of [3H] -BK and BK causes excitation of nociceptors (5). Some of the information about the stimulus transduced by activation of that is nociceptors is converted to a form accesible to the brain and is transferred to the dorsal horn of the spinal cord. In addition, some of the information is transmitted to the periphery through axon reflex phenomena: that is, primary afferent fibers serve efferent as well as afferent functions. With to regard the former function, recent pathophysiological studies in humans and laboratory animals have indicated that primary afferent fibers containing neuropeptides such as tachykinin and calcitonin gene-related peptide (CGRP) may contribute to the pathogeneses of thermal injury, polyarthritis, respiratory-tract irritation, etc. (6-9). In this connection, in the skin of the rat hind paw, we observed that noxious heat stimulation led to the releases of substance P (SP) and CGRP from the small-diameter afferent fibers and that these neuropeptides released in the periphery are closely related to edema formation in the thermal injury reaction (10,ll). These findings suggest that nociceptor activity is not merely a passive signal indicating that tissue damage has occurred, but may also play an active role in body defense by participating in the inflammatory process; that is, afferent impulses from nociceptors generated by a noxious stimulus cause release of neuropeptides, which finally control the inflammatory response. aim of this study was to demonstrate more clearly the The peripheral involvement of kinins in the SP released from the noxious heat afferent fibers following endings of primary weight (HMW) stimulation. For this, we used the high molecular strain rat low molecular weight (LMW) kininogen-deficient and normal rat strain Norway-Katholiek (B/N-Ka)) and the (Brown (Brown Norway-Kitasato (B/N-Ki)), and determined the releases of extravasation and BK into the S.C. perfusate and Evans blue SP evoked by noxious heat stimulation.
Materials
and Methods
rats were obtained and B/N-Ki B/N-Ka Kitasato School of Medicine, laboratory of strains were bred and maintained in the animal faculty, Osaka University.
animal from the Both University. laboratory of our
with urethane (780 B/N-Ka and B/N-Ki rats were anesthetized introduced A double coaxial polyethylene tube was i.p.). the containing saline into the S.C. space of the instep, and and the bestatin (3 mg/lOO inhibitor aminopeptidase ml)
mg/kg
Vol.56,No.20,1995
SubstancePReleaseandBradykinin
1681
dipeptidyl inhibitor, captopril carboxypeptidase (0.1 mM) was perfused through this tube with a peristaltic pump at a rate of 0.1 ml/min. The perfusate was collected through the outer tube in lo-min fractions in test tubes in an ice bath using a fraction collector. The samples were lyophilized and then radioimmunoassayed for BK and SP using the corresponding antisera. Details of the radioimmunoassay procedure of SP were as described previously (12). For the assay of BK, the perfusate was collected in test tubes containing two volumes of ethanol in an ice bath. The mixture was centrifuged at 3400 g for 15 min and 2.5 ml of supernatant was lyophilized. After lyophilization, each lo-min fraction was redissolved in 200 1-r1 of phosphate buffered (10 m&l, pH 7.4) containing 0.1 % gelatin, and saline then 10000 100 111 of [3H]BK, adjusted to approximately cpm per tube, and 100 1~1 of anti-BK rabbit serum (dilution 1 in 15000) were successively added. The polyethylene test tubes containing 4 OC for 12-18 h. sample solution were incubated at For separation of bound and free radiolabeled peptides, 250 1~1 of 1 % of dextran-coated charcoal was added to each solution tube and incubation continued at 4'C for 30 min. Thereafter, was the 15000 g for 20 min at 4 OC. tubes were centrifuged at The 650 ~1 of the supernatant was measured in a radioactivity of liquid scintillation counter. The antiserum of BK showed crossreactivities IleLys-BK, of 68 %, 60 % and less than 1 90with Ser-BK and SP, respectively. For measurement of vascular permeability, the S-C. space of perfused as described above. Evans blue the instep was (50 urethane mg/kg) was injected i.v. into animals anesthetized with side (780 mg/kg i.p.), and 20 min later the paw on the perfused The amount of immersed for 20 min in a water-bath at 47'C. was released into the perfusate (collected in lo-min fractions) dye a wavelengh 620 nm with was measured at a spectrophotometer Shimadzu Scientific Instruments, Kyoto, Japan). (model W-240, are shown as mean I_ SEM for the number of animals Results indicated. statistical analysis Student's t-test for For P values of unpaired values was less than 0.05 were used. considered to be significant. Materials: The following drugs were used: BK and SP from Peptide Institute (Japan), BK antiserum (a gift from Nippon Zoki Pharmaceutical co. (Hyogo, Japan)), and [125I][Tyr8]SP and [2,3prolyl-3,43H(N)]-BK from DuPont/New England Nuclear Research Products (USA).
Results Effect of heat stimulation and B/N-Ki rats
an
on the release
of Evans
blue
in B/N-Ka
As can be seen in Fig. 1, noxious heat stimulation caused increase in Evans blue release in the both B/N-Ki rats and
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Vol. 56, No. 20, 1995
Substance P Release and Bradykinin
B/N-Ka
rats, but the stimulus-evoked Evans blue release, was than in the significantly less in the B/N-Ka strain BIN-Ki strain: each amount of Evans blue released in the two 10 min fractions during stimulation in B/N-Ki (n=lO) and B/N-Ka rats (n=18) were 2.81 ?r 0.1 i_rg/kg (first lo-min samples) and 3.92 + 0.13 pg/kg (second lo-min samples), and 0.92 + O.ll.rg/kg (first lo-min lo-min samples) and 2.25 + 0.12 pg/kg (second samples), respectively. Local application of BK (lo-4 M) to the S.C. perfusate of the B/N-Ka strain significantly enhanced the stimulus-evoked Evans blue release, the maximum increase being almost the same as that in the B/N-Ki strain (4.12 f 0.1 I-lglkg, n=15).
o--O BIN-Ki O--O BIN-Ka
I
H
E/N-Ka
(BK 10X4
5 t
OO-
1
2
3 -(hr)
heating EK (10-M)
Fig.
1
Effect of heat stimulation on the release of Evans instep of blue into the S.C. perfusate in the hind and effect of local B/N-Ki and B/N-Ka rats, B/N-Ka BK to the S.C. perfusate of application of rats. Values are means of at least 10 experiments. range of The deviation of each value is within the value each symbol. * P co.05 when compared with the obtained in the B/N-Ka rats at each time. Effect rats
of heat
stimulation
on the releases
of SP and BK in B/N-Ki
As shown in Fig. 2(A), BK release did not increase in the markedly for lo-20 min but increased first lo-min samples, (second lo-min samples) after onset of immersion of the hind paw in hot water at 47'C. On the other hand, heat stimulation for 20 time: that is, a significant increase of SP with min led to marked release of SP was detected immediately after the start of
Vol. 56, No. 20, 1995
noxious rapidly
1683
Substance P Release and Bradykinin
heat stimulation and this stimulus-evoked SP release decreased after the end of stimulation (Fig. 2(B) ).
was
(A)
1oll~ L
.9
JO 60
120
160
(mid
120
180
(mid
heating
(B) 2000 0
z
2
m, 1500-
a :
5
lOOO-
; 4 n ap
500-
lo;-
Q 60
1 heating
Fig.
2
Effect of noxious heat stimulation on the releases of BK (A) and SP (B) into the S.C. perfusate .in the hind instep of BIN-Ki rats, and change in the subcutaneous noxious For temperature (upper line in (A)). stimulation, a rat paw was immersed in a water-bath change in For calculating the at 47OC for 20 min. the four release, the mean value for peptide the fractions collected before heating was taken as control value (100%). The peptide contents in each successive fraction are expressed as percentages of Each value represents the mean f this control value. SEM of 16 experiments. Release of BK induced Ka rats As
shown
by heating
in Figs.
the hind paw of B/N-Ki
2 and 3, immersion
of the hind
and
paw
B/N-
of
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Substance P Release and Bradyldnin
Vol. 56, No. 20, 1995
B/N-Ki rats in hot water caused a significant increase of BK in while similar treatment of B/N-Ka the perfusate, rats caused significantly less increase of BK. The average amounts of BK released in the two fractions obtained in the 20-min collection periods before and during noxious heat stimulation were, respectively, 0.124 + 0.015 pmol/lO min and 0.430 + 0.06 pmol/lO min in B/N-Ki rats, and 0.008 f 0.002 (n=15) pmol/lO min and 0.012 + 0.002 pmol/lO min (n=16) in B/N-Ka rats. Release of SP induced Ka rats
by heating
the hind paw of B/N-Ki
and
B/N-
The SP levels in the resting state; that is, the average amount of SP released in the fractions in the two 20-min collection noxious period before heat stimulation were 0.017 f 0.004 pmol/lO min (n=lO) in B/N-Ki rats and 0.019 f 0.003 fmol/lO min (n=lO) in B/N-Ka rats. As shown in Fig. 4, noxious heat stimulation produced a marked increase of SP release in both strains, but the SP level in the B/N-Ka strain (0.069 + 0.014 pmol/lO min, n=lO) was significantly lower than that in the B/NKi satrain (0.121 + 0.014 pmol/lO min, n=lO). To determine whether release of SP into the subcutaneous perfusate evoked by heat stimulation was modulated by BK, we infused BK into the foot on the side of perfusion through an arterial cannula inserted into the contralateral iliac common artery of B/N-Ka rats. At the concentration employed BK enhanced the heat-evoked SP release to almost (10 umol/kg), the same level as that in B/N-Ki rats after heat stimulation without BK treatment.
Discussion plasma Recent study on rat kininogen demonstrated that rat and kallikrein releases BK from high molecular weight kininogen, from high and low kallikrein releases also BK rat urinary there is no In addition, weight kininogens molecular (13). Taking sequence in rat kininogens by cDNA study (14). kallidin showed these findings into account, although the antiserum of BK Ile-Ser-BK, of 68 % and 60 % with Lys-BK and cross-reactivities that a large be reasonable to consider respectively, it may portion of kinins detected in the perfusate is BK. As shown in Fig. 2A, an increase in BK release was observed in the second lo-min samples, i.e. 10 - 20 min after the start of first immersion of the hind paw in water at 47"C, but not in the observation This result was consistent with the lo-min samples. that the showed Sato and Yamaguchi Starr and West (15). of release enzyme and kininase increase of kininogen, kinin-forming heat onset of behind the also lagged perfusate into the On the other hand, a marked increase in SP stimulation (16). heat start of after the immediately detected release was stimulation and this stimulus-evoked SP release rapidly decreased the difference between The stimulation. the end of after
Vol.56,No.U), 199.5
Fig.
Substance P Release and Bradykinin
1685
3 0.6L
Effects of noxious heat stimulation on the releases of BK into the S.C. perfusate in the hind instep of B/N-Ki and B/N-Ka rats. Each value represents the mean + SEM for BK release in the two fractions obtained in 20-min collection periods before, during and after heat stimulation (stim.), in at least 15 experiments.
0
E/N-Ki
Q
B/N-Ka
PCO.05
I
I
OS”
2 0.4,a .c .G 5 0.3Ti m 2 0.2-
0.1-
BIN-Ki
before
during
after
stim.
stim.
stim.
B/N-Ka BIN-Ka (BK lO_‘mol/kg
before
stim.
during stim.
Fig.
i.a.)
after stim.
4
Effect of noxious heat stimulation on the release of SP into the S.C. perfusate in the hind instep of B/NKi and B/N-Ka rats, and influence of BK infused into artery of B/N-Ka rats on its the common iliac release. Each value represents the mean f SEM for SP obtained in release in the two fractions 20-min collection periods before, during and after heat stimulation (stim.), in 10 experiments. release of patterns of BK and SP may be attributed to the difference between the responses to heat stimulation of these
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Substance P Release and Bradykink
Vol. 56,No. 2Ql995
It is known that BK is released peptide-releasing systems. from called kininogens - by a precursor in plasma - a2-globulins the action of proteolytic enzymes with the specificity of trypsin we have demonstrated that SP into the released (171, whereas subcutaneous perfusate following heat stimulation originates mainly from the peripheral endings of capsaicin-sensitive, smalldiameter afferent fibers (10). the B/N strain of rats were Two types of used in the present study, the mutant rat strain named Brown Norway Katholiek (B/N-Ka) and the normal strain, Brown Norway Kitasato (B/NKi) (18,19). The former is deficient in high and low molecular weight kininogens in the plasma, and the level of prekallikrein is only about 30 % of the latter (20). In the present study, the amounts of BK released into the perfusate both before and during noxious heat stimulation were much lower in B/N-Ka rats than in B/N-Ki rats. In addition, B/N-Ka rats showed significantly less heat-evoked extravasation than B/N-Ki rats, but the application of BK to the S.C. perfusate of B/N-Ka rats increased heat-evoked extravasation to almost the level observed in B/N-Ki rats. These results indicate that BK detected in the extravascular space may be largely produced through activation of the plasma kallikreinkinin system, and this plasma kinin system has an important role in the thermal injury reaction. Concerning the functional interaction between BK and SP released by noxious heat stimulation, the amount of the heatwas significantly lower in the evoked SP release kininogenstrain B/N-Ka, than in B/N-Ki rats. deficient Furthermore, as shown in Fig. 4, infusion of BK into the common iliac artery of rats, as in the extravasation experiment (Fig. l), B/N-Ka enhanced the level of the heat-evoked SP release to nearly the that in normal rats. findings suggest the level of These contribution of the plasma kinin system to SP release induced by been In this connection, it is noteworthy that BK has heating. also shown to act not only as a powerful pain-inducing agent but topical In fact, as an important mediator in inflammation (21). administration of BK increases blood flow and vascular permeabilperipheral tissues including the skin (4) and ity in most by and at least part of the response is mediated airways (221, For small-diameter afferent fibers capsaicin-sensitive, (23). evoked by BK instilled into guineaexample, broncho-constriction can be abolished by capsaicin pre-treatment (24). airways pig Ul\ caused % of the extravasation almost 60 Furthermore, topically into the nose of rats was inhibited b;' CPinstilled 96345, a selective inhibitor of NKl receptors (25). BK receptors have been localized to nociceptive pathways (peripheral terminals small small-diameter afferent fibers and of capsaicin-sensitive, cells in dorsal root ganglia) by autoradiography of L3~l-~~ (5). saphenous nerve the release of SP from evoked Moreover, BK that neuromas of the rat (26). We also have already demonstrated heating induced by of SP and the edema formation release the pretreatment simultaneous significantly by the inhibited were bromelain (which causes a specific depletion of HMW with stem kininogen) and emorfazone (a nonacidic antiinflammatory drug that
Vol.56,No.20,1995
Substance P Release andBradylcinin
1687
inhibits the release of BK into extravascular space (16)), and by the intra-arterial infusion of Des-Arg9-[Leu8]BK (BK antagonist) These observations and our present results strongly (10,271. the hypothesis that BK released into extravascular support the noxious heat stimulation causes excitation of BK space by receptors in sensory nerve terminals that, in turn, activates capsaicin-sensitive, small-diameter afferent fibers to release SP from peripheral endings through a mechanism such as an axonreflex arrangement. In the present experiment, noxious heat stimulation of B/NKa rats induced a very low level of BK release, but a significant increase in SP release. Furthermore, in the first lo-min sample immediately from normal rats, a large release of SP was detected after the start of the experiment without BK release, suggesting substances other than BK may be involved in SP that chemical especially in its initial stage of release, evoked by release, the peripheral endings of noxious heat stimulation from capsaicin-sensitive primary afferent fibers.
Acknowledgements
their
We would generous
like to thank Drs. Yasuo Imai and Sou Takiuchi assistance with this project.
for
References 1. 2. 3. 4. 5.
6.
7. 8. 9. 10. 11. 12. 13. 14. 15.
G.P. LEWIS, Handb. exp. Pharmacol. 25 516-530 (1970) STARR and G.B. WEST, Brit. JF Pharmacol. 178-187 M.S. -31 (1967) D. ARMSTRONG, Handb. exp. Pharmacol. 25 434-481 (1970) Z. KHALIL and R.D. HELME, Brain Res. 589 102-108 (1992) L.R. STERANKA, D.C. MANNING, C.J. DEHAAS, J.W. FERKANY, S.A. CONNOR, R.J. VAVREK, J.M. STEWART and S.H. BOROSKY, J.R. SNYDER, Proc. Natl. Acad. Sci. USA, 85 3245-3249 (1988) F. LEMBECK, Substance P: metabolism and biological actions, C.C. Jordam and P. Oehme (Eds), 137-151, Taylor and Francis, London (1985) X.-Y. HUA, Acta Physiol. Stand. Suppl. 551 1-45 (1986) C.A. MAGGI and A. MELI, Gen. Pharmacol. 19 l-43 (1988) K.A. SHARKEY, Ann. N. Y. Acad. Sci. 664 425-442 (1992) N. YONEHARA, T. SHIBUTANI and R. INOKI, J. Pharmacol. Exp. Ther. 242 1071-1076 (1988) N. YONEHARA, Y. IMAI and R. INOKI, Japan. J. Pharmacol. 56 381-384 (1991) N. YONEHARA, T. SHIBUTANI, H.-Y. TSAI and R. INOKI, Eur. J. Pharmacol. 129 209-216 (1986) H. KATO, K. ENJYOJI, T. MIYATA, I. HAYASHI and S. OH-ISHI, Biochem. Biophys. Res. Commun. 127 289-295 (1985) S. FURUYO-KATO, A. MATSUMOTO, N. KITAMURA and S. NAKANISHI, J. Biol. Chem. 260 12054-12059 (1985) M.S. STARR and G.B. WEST, Brit. J. Pharmacol. 31 178-187 (1967)
1688
16.
17. 18. 19. 20. 21. 22. 23. 24. 25.
26. 27.
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Vol. 56, No. 20, 1995
Res. -32 379M. SAT0 and A. YAMAGUCHI, Arzneim-Forsch./Drug 382 (1982) ROSENFELD, Am. J. ROCHA E SILVA, W.T. BERALDO and G. M. Physiol. 156 261-273 (1949) S. OH-ISHI, I. HAYASHI, K. SATOH and T. NAKANO, Thrombosis Res. 33 371-377 (1984) I. HAZSHI, S. OH-ISHI, H. KATO, K. ENJYOGI, S. IWANAGA and T. NAKANO, Thrombosis Res. 39 313-321 (1985) S. OH-ISHI, K. SATOH, I. HAYASHI, K. YAMAZAKI and T. NAKANO, Thrombosis Res. 28 143-147 (1982) D. REGOLI and J.>ARABE, Pharmacol. Rev. 32 1-46 (1980) J.O. LOTVALL, K. TOKUYAMA, P.J. BARNES and K.F. CHUNG, Eur. J. Pharmacol. 200 211-217 (1991) JANCSG, E. KItiLY and A. JANCSG-GABOR, Int. J. Tissue G. React. 2 57-66 (1980) M. ICHINOSE, M.G. BELVISI and P.J. BARNES, J. Pharmacol. Exp. Ther. 253 594-599 (1990) GEPPETTI, J. BAKER, G. PETERSSON, G. BERTRAND, P. C. 2456-2461 J.A. NADEL, J. Appl. Physiol. PIEDIMONTE and -74 (1993) Lett. 108-113 WHITE and M. ZIMMERMANN, Neurosci. D.M. -92 (1988) N. YONEHARA, Y. IMAI, T. SHIBUTANI and R. INOKI, KININS V, Fujii 529-534, PART B, K. Abe, H. Moriya and S. (Eds), Plenum Publishing, New York (1989)