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Tachykinin receptor subtype that mediates the increase in vascular permeability in guinea pig skin
Pergamon Press
Life Sciences, Vol. 44, pp. 1089-1095 Printed in the U.S.A.
TACHYKININ RECEPTOR SUBTYPE THAT MEDIATES THE INCREASE IN VASCULAR PERMEABILITY IN GUINEA PIG SKIN
Itsuo
Iwamoto and Jay A. Nadel
Cardiovascular Research Institute and Departments of Medicine and Physiology University of California, San Francisco San Francisco, CA 94143-0130 (Received in final form February 13, 1989) Summary To determine the tachykinin receptor subtype that mediates the increase in vascular permeability, we examined the rank order of potency of tachykinins for inducing plasma extravasation in guinea pig skin and the specificity of tachykinin-induced tachyphylaxis of the responses. Plasma extravasation of the skin induced by tachykinins was NK-1 (SP-P)-type response from the rank order of potency of mammalian and nonmammalian tachykinins. Tachyphylaxis of the vascular response was induced by intradermal preinjection of mammalian tachykinins and was tachykinin-specific. In substance P (SP) tachyphylaxis (where SP was preinjected), the response to SP, not to neurokinin A (NKA) or neurokinin B (NKB), was decreased. In NKA tachyphylaxis and NKB tachyphylaxis, the response to NKA, not to SP or NKB, and the response to NKB, not to SP or NKA, were decreased, respectively. Thus, we conclude that the apparent NK-l-type response is mediated through three mammalian tachykinin receptors, NK-1, NK-2, and NK-3, which are specifically stimulated by their preferred agonist, SP, NKA, and NKB, respectively.
Increasing evidence suggests that mammalian tachykinins substance P (SP) and neurokinin A (NKA) (possibly also neurokinin B; NKB) are major mediators of neurogenic plasma extravasation. Neurogenic inflammation induced by antidromic nerve stimulation occurs in the skin and causes an increase in vascular permeability [ll. The inflammation is inhibited by pretreatment with capsaicin, which depletes sensory neurons of SP 121, and by SP antagonists [3l. Furthermore, the three tachykinins, SP, NKA, and NKB, increase vascular permeability in the skin when administered intradermally [4,51. In addition, three subtypes of tachykinin receptors, NK-1, NK-2, and NK-3, their preferred agonists of which are SP, NKA, and NKB, respectively, have recently been identified by pharmacological studies and receptor binding assays 16-91. However, the type of tachykinin receptor responsible for mediating the increase in vascular permeability remains unknown.
In this study, to determine the tachykinin receptor subtype that mediates the increase in vascular permeability, we examined the relative potencies and rank order of mammalian and nonmammalian tachykinins for inducing plasma
0024-3205189
$3.00
+ .OO
Copyright (c) 1989 Pergamon Press plc
1090
Tachykinin and Vascular Permeability
Vol. 44, No. 16, 1989
extravasation in guinea pig skin, and we determined the specificity of tachykinin-induced tachyphylaxis of the responses. Methods 1.
Plasma extravasation in guinea pig skin
Hartley male guinea pigs (450-500 g) were anesthesized with intraperitoneal pentobarbital (25 mg/kg), and the dorsal skin was shaved. Five minutes before intradermal injections of tachykinins, Evans blue dye (30 mg/kg) was administered intravenously. Tachykinins (50 ul) were then injected intradermally in the shaved dorsal skin. We injected tachykinins at six to eight sites of the dorsal shaved skin in an animal. Twenty minutes after the injection of tachykinins, Evans blue extravasation was evaluated by calculating the circular area of each Evans blue spot from the mean value of two perpendicular diameters (the largest diameter and the diameter at right angles to this) of the spot [51. Evans blue extravasation at a given concentration of a tachykinin was evaluated in duplicate using one animal, and the number used to determine the mean value is indicated in the figure legends. As a control, 10 mM phosphate-buffered saline (PBS; pH 7.4; 50 ~1) was injected intradermally and did not induce any significant Evans blue extravasation. 2.
Rank order of potency of tachykinins for inducing plasma extravasation
To determine the tachykinin receptor sybtype responsible for increasing vascular permeability in the skin, we determined the rank order of potency of mansnalian and nonmammalian tachykinins (10-g to 10-5 M) for inducing plasma extravasation in guinea pig skin from the concentration-response curves. To assess the C-terminal and N-terminal dependency of tachykinins in the response, we also determined the potency of the fragments SPg-11 and SPl-g. 3.
Tachykinin-induced tachyphylaxis and its specificity
To determine whether mammalian tachykinin-induced plasma extravasation is mediated solely through NK-1 (SP-P) receptors or through multiple tachykinin receptors specific for individual tachykinins, we induced tachyphylaxis of the vascular responses by intradermal preinjection of each of the three mammalian tachykinins and examined the specificity of the tachyphylaxis. First, one of the tachykinins (50 I.il) or PBS (50 ul) was injected intradermally. Thirty minutes after the first injection, another injection of the same or a different tachykinin (50 ~1) was performed at the same injection site. Evans blue was administered 5 min before the second injection. No Evans blue extravasation was observed when the dye was administered more than 20 min after the first injection of tachykinins. The specificity of the tachyphylaxis was determined by using equipotent concentrations of tachykinins (3 x 10-7 M SP, 10-5 M NKB, and 2 x 10-5 M NKA). 4.
Drugs used
Substance P and Evans blue were purchased from Sigma Chemical Co. (St. Louis, MO). Neurokinin A, neurokinin B, physalaemin, eledoisin, kassinin, SPB-11, and SPl-g were purchased from Peninsula Laboratories (Belmont, CA).
Vol.
44, No.
16,
1989
Tachykinin
and Vascular
Permeability
1091
All of the tachykinins with the exception of NKB (2 x 10-3 M) and SP fragments were dissolved in 0.1 N acetic acid and then diluted with PBS. Neurokinin B (2 x 10-2 M) was dissolved in 10 N acetic acid and then diluted with PBS. Evans blue was dissolved in physiological saline.
5.
Data analysis
Data are reported as means l SD. statistical comparison of the data.
The paired Student's test was used for
Results 1.
Potency and rank order of mannnalian and nonmamnalian tachykinins for inducing plasma extravasation in guinea pig skin
Mammalian and nonmarnmalian tachykinins (10-g to 10-S M) induced plasma extravasation in guinea pig skin concentration-dependently when administered intradermally (fig. 1). Among mammalian tachykinins, the rank order of potency for inducing plasma extravasation in guinea pig skin was SP>NKB?.NKA (fig. 1, upper). The difference in the potency between SP and NKB (or NKA) was about a half log. Among nonmammalian tachykinins, the responses were equipotent (physalaemin?eledoisin=kassinin) (fig. 1, lower). These findings syggest that the tachykinin-induced plasma extravasation in guinea pig skin is an NK-1 (SPP)-type response [61.
SP6-11 (10-6 to 10-S M) induced plasma extravasation concentrationdependently (fig. 1, lower), and the potency was one log lower than SP. SPI-g (up to 10-5 M) did not induce plasma extravasation. These findings suggest that C-terminal hexapeptide of SP is important in increasing vascular permeability. 2.
Mammalian tachykinin-induced tachyphylaxis of the responses
Substance P, NKB, and NKA induced tachyphylaxis of the vascular responses concentration-dependently. Intradermal preinjection of SP, NKB, or NKA (10-8 to 10-5 M) decreased plasma extravasation induced by the second injection of the same tachykinin (10-5 M) depending on the concentration of tachykinin preinjected (fig. 2, upper). The maximum degree of tachyphylaxis that was produced, that is, 35% to 38% reduction in the responses induced by preinjections of 10-8 M tachykinins, correspond to approximately 90% inhibition of the responses from the concentration-response curves (fig. 1, upper). Tachyphylaxis of the vascular response induced by mansnalian tachykinins was tachykinin-specific. In SP tachyphylaxis (where SP was preinjected intradermally), only the response to SP was decreased, but the responses to NKB or NKA were the same as controls (preinjection of PBS) (fig. 2, lower). In NKB tachyphylaxis and NKA tachyphylaxis, only the responses to NKB and NKA, respectively, were decreased, but the responses to SP or NKA, and the responses to SP or NKB, were the same as controls, respectively. The results suggest that SP induced the response solely through NK-1 (SP-P) receptors, NKA solely through NK-2 (NKA) receptors, and NKB solely through NK-3 (NKB) receptors.
1092
Tachykinin and Vascular Permeability
Vol. 44, No. 16, 1989
Evans blue extravasation
80604020-
80 60
-9
-8 -7 Tachykinins
FIG.
-6 (log M)
-5
1.
mammalian (upper) and curves of Concentration-response nonmammalian (lower) tachykinin-induced plasma extravasation in guinea pig skin. Evans blue extravasation was measured 20 min after intradermal injection of 50 cjlof a tachykinin. 0 = SP, I = NKB, and A = NKA in the upper panel. 0 = physalaemin, D = eledosin, A = kassinin, 0 = SPg_11, and V = SPl-g in the lower panel. Data are means + SD for six experiments in the upper*panel and for three P< 0.025, significantly experiments in the lower panel. *PC 0.05, different from the mean of the response induced by NKB or NKA.
Tachykinin and Vascular Permeability
Vol. 44, No. 16, 1989
1093
Evans blue extravasation
-6 -7 -6
-6 -7 -6 SP
(log
SP
M)
NKB
(log
NKB
-6 -7 -6 M)
NKA
(log
M)
NKA
FIG. 2. Concentration-dependency (upper) and specificity (lower) of tachyphylaxis of mammalian tachykinin-induced plasma extravasation in guinea pig skin. Thirty minutes after the first intradermal injection of a tachykinin, the second injection was performed at the original injection site. Five minutes before the second injection, Evans blue was administered intravenously. In the upper panel, the concentrations of tachykinins used in the first injections were 10-6 to 10-6 M, and the second injection was made with the same tachykinin (10-6 M). In the lower panel, the equipotent concentrations of tachykinins used were the same in the first and second injections (3 x 10-T M SP, 10-6 M NK6, and 2 x 104 M NKA). The axis is normalized to the values that were obtained when the first injection was made with PBS (control). Oata are means * SO for four experiments in the upper panel and five experiments in the lower panel. lP c 0.025, **P c 0.005, significantly different from the mean of the control.
1094
Tachykinin and Vascular Permeability
vol. 44, No. 16, 1989
Discussion
In this study, we show that three mammlian tachykinins, SP, NKA, and NKB, increase vascular permeability in guinea pig skin through three different subtypes of tachykinin receptors, i.e., NK-1 (SP-P) receptors, NK-2 (NKA) receptors, and NK-3 (NKB) receptors, and that each tachykinin preferentially stimulates its specific receptors in the vascular response, i.e., SP, NKA, and NKB stimulate NK-1, NK-2, and NK-3 receptors, respectively. On the basis of the rank order of potency of mammalian and nonmammalian tachykinins for inducing plasma extravasation in guinea pig skin, we found that the vascular response was NK-l-type (fig. 1) [61. The vascular response was not inhibited by a histamine HI antagonist and seemed to be direct effects of tachykinins [lo]. We also found that the vascular response was C-terminal hexapeptidedependent (fig. 1). Then, to determine whether the apparent NK-l-type response is mediated solely through NK-1 receptors or through multiple tachykinin receptors, we induced tachyphylaxis of the response to each of the three mammalian tachykinins and determined the specificity. We found that the tachphylaxis was specific for each tachykinin and did not affect the responses to the other two tachykinins (fig. 2). Thus, the apparent NK-1 (SP-P)-type response of plasma extravasation in the skin is mediated through three different subtypes of tachykinin receptors. We interpret the fact that the responses induced by the three mammalian tachykinins are nearly equal as suggesting that the responses are mediated via three different tachykinin receptors. If the responses were mediated solely through NK-1 (SP-P) receptors, the responses to NKA and NKB should have been much less than the response to SP, because NK-1 receptors have tens to hundreds of times less For the same reason Clll, if the response to SP affinity for NKA and NKB [91. was mediated mainly through NK-2 (NKA) receptors, the response to SP should have been hundreds of times less than the response to NKA. Because plasma extravasation is caused mainly by contraction of endothelial cells of capillaries and venules [121, our findings can be interpreted in terms of three different tachykinin receptors located in endothelial cells of cutaneous blood vessels. Indeed, an autoradiographic study showed SP binding sites in the endothelium of the blood vessels [131. Our findings are in close agreement with the finding that the potency of NKB and NKA for releasing arterial relaxing factor(s) from the endothelium of rabbits was one-fourth to one-fifth of that of SP [141. Thus, the possible existence of three different tachykinin receptors in the endothelium of cutaneous blood vessels suggests that all three endogenous tachykinins may play a physiological role in increasing vascular permeability in the skin. In this regard, it is interesting that SP and NKA are co-localized in certain primary afferent neurones, whereas NKB is localized in interneurones or ascending pathways rather than in primary afferents [151. Therefore, we speculate that each tachykinin may be released to increase vascular permeability from different neurones, probably by different stimuli. Finally, the precise mechanism of the tachyphylaxis in tachykinin-induced plasma extravasation is unknown. However, the tachyphylaxis is a homologoustype desensitization, not a heterologous one, so tachykinin receptors themselves are most likely involved in the phenomenon, and phosphorylation and/or internalization of the receptors might be responsible for inducing the unresponsiveness of the receptor-mediated pathways as indicated in other experimental systems [161. In summary, we have shown that SP, NKA, and NKB induce plasma extravasation in guinea pig skin specifically through NK-1, NK-2, and NK-3 receptors, respectively.
Vol. 44, No. 16, 1989
Tachykinin and Vascular Permeability
1095
Acknowledgments We thank Ms. I. Ueki for technical assistance and Ms. B. Cost and Ms. P. Snell for assistance in preparing the manuscript. This study was supported in part by NIH Program Project Grant HL-24136. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
B. PERNOW, J. Immunol., 135 812S-815s (1985). F. LEMBECK and P. HOLZEcNaunyn Schmiedebergs Arch. Pharmacol., -310 175-183 (1979). F. LEMBECK, J. OONNERER and L. BARTHO, Eur. J. Pharmacol., -85 171-176 (1982). J.C. FOREMAN, C.C. JORDAN, P. OEHME and H. RENNER, J. Physiol. (Lond.), 335 449 (1983). V;-OEVILLIER, 0. REGOLI, A. ASSERAF, B. OESCOURS, J. MARSAC and M. RENOUX, Pharmacology 32 340 (1986). L.L. IVERSEN, M.R. HARLEY, B.E.B. SANDBERG, C.M. LEE, R.O. PINNOCK and S.P. WATSON, Ciba Found. Symp., 91 186-195 (1982). R. LAUFER, U. WORMSER, Z.Y. FRImMAN, C. GILON, M. CHOREV and Z. SELINGER, Proc. Natl. Acad. Sci. USA, 82 7444-7448 (1985). M.A. CASCIERI and T. LIANG, Life Sci.,T5 179-184 (1984). S.H. BUCK, E. BURCHER, C.W. SHULTS, W.LOVENBERG and T.L. O'OONOHUE, Science, 226 987-989 (1984). I. IWAMOK I.F. UEKI, O.B. BORSON and J.A. NAOEL, Int. Arch. Allergy Appl. Immunol., (in press). Y. MASU, K. NAKAYAMA, H. TAMAKI, Y. HARAOA, M. KUNO and S. NAKANISHI, Nature, 329 836-838 (1987). G. MAJNO3.M. SHEA and M. LEVENTHAL, J. Cell Biol., 42 647 (1969). J.A. STEPHENSON, R.J. SUMMERS and E. BURCHER, Eur. J.-Pharmacol., -142 391
:3: 14. 15. 16.
(?87). O'ORLEANS-JUSTE, S. OION, G. ORAPEAU and 0. REGOLI, Eur. J. 125 37 (1985). Ph&macol T. OGAWA,';.mNAZAWA and S. KIMURA, Brain Res., 359 152 (1985). L.M.F. LEEB-LUNDBERG, S. COTECCHIA, J.W. L0MASNEcJ.F. OEBERNAROS, R.F. LEFKOWITZ and M.G. CARON, Proc. Natl. Acad. Sci. USA, -82 5651-5655 (1985).
Life Sciences, Vol. 44, pp. 1089-1095 Printed in the U.S.A.
TACHYKININ RECEPTOR SUBTYPE THAT MEDIATES THE INCREASE IN VASCULAR PERMEABILITY IN GUINEA PIG SKIN
Itsuo
Iwamoto and Jay A. Nadel
Cardiovascular Research Institute and Departments of Medicine and Physiology University of California, San Francisco San Francisco, CA 94143-0130 (Received in final form February 13, 1989) Summary To determine the tachykinin receptor subtype that mediates the increase in vascular permeability, we examined the rank order of potency of tachykinins for inducing plasma extravasation in guinea pig skin and the specificity of tachykinin-induced tachyphylaxis of the responses. Plasma extravasation of the skin induced by tachykinins was NK-1 (SP-P)-type response from the rank order of potency of mammalian and nonmammalian tachykinins. Tachyphylaxis of the vascular response was induced by intradermal preinjection of mammalian tachykinins and was tachykinin-specific. In substance P (SP) tachyphylaxis (where SP was preinjected), the response to SP, not to neurokinin A (NKA) or neurokinin B (NKB), was decreased. In NKA tachyphylaxis and NKB tachyphylaxis, the response to NKA, not to SP or NKB, and the response to NKB, not to SP or NKA, were decreased, respectively. Thus, we conclude that the apparent NK-l-type response is mediated through three mammalian tachykinin receptors, NK-1, NK-2, and NK-3, which are specifically stimulated by their preferred agonist, SP, NKA, and NKB, respectively.
Increasing evidence suggests that mammalian tachykinins substance P (SP) and neurokinin A (NKA) (possibly also neurokinin B; NKB) are major mediators of neurogenic plasma extravasation. Neurogenic inflammation induced by antidromic nerve stimulation occurs in the skin and causes an increase in vascular permeability [ll. The inflammation is inhibited by pretreatment with capsaicin, which depletes sensory neurons of SP 121, and by SP antagonists [3l. Furthermore, the three tachykinins, SP, NKA, and NKB, increase vascular permeability in the skin when administered intradermally [4,51. In addition, three subtypes of tachykinin receptors, NK-1, NK-2, and NK-3, their preferred agonists of which are SP, NKA, and NKB, respectively, have recently been identified by pharmacological studies and receptor binding assays 16-91. However, the type of tachykinin receptor responsible for mediating the increase in vascular permeability remains unknown.
In this study, to determine the tachykinin receptor subtype that mediates the increase in vascular permeability, we examined the relative potencies and rank order of mammalian and nonmammalian tachykinins for inducing plasma
0024-3205189
$3.00
+ .OO
Copyright (c) 1989 Pergamon Press plc
1090
Tachykinin and Vascular Permeability
Vol. 44, No. 16, 1989
extravasation in guinea pig skin, and we determined the specificity of tachykinin-induced tachyphylaxis of the responses. Methods 1.
Plasma extravasation in guinea pig skin
Hartley male guinea pigs (450-500 g) were anesthesized with intraperitoneal pentobarbital (25 mg/kg), and the dorsal skin was shaved. Five minutes before intradermal injections of tachykinins, Evans blue dye (30 mg/kg) was administered intravenously. Tachykinins (50 ul) were then injected intradermally in the shaved dorsal skin. We injected tachykinins at six to eight sites of the dorsal shaved skin in an animal. Twenty minutes after the injection of tachykinins, Evans blue extravasation was evaluated by calculating the circular area of each Evans blue spot from the mean value of two perpendicular diameters (the largest diameter and the diameter at right angles to this) of the spot [51. Evans blue extravasation at a given concentration of a tachykinin was evaluated in duplicate using one animal, and the number used to determine the mean value is indicated in the figure legends. As a control, 10 mM phosphate-buffered saline (PBS; pH 7.4; 50 ~1) was injected intradermally and did not induce any significant Evans blue extravasation. 2.
Rank order of potency of tachykinins for inducing plasma extravasation
To determine the tachykinin receptor sybtype responsible for increasing vascular permeability in the skin, we determined the rank order of potency of mansnalian and nonmammalian tachykinins (10-g to 10-5 M) for inducing plasma extravasation in guinea pig skin from the concentration-response curves. To assess the C-terminal and N-terminal dependency of tachykinins in the response, we also determined the potency of the fragments SPg-11 and SPl-g. 3.
Tachykinin-induced tachyphylaxis and its specificity
To determine whether mammalian tachykinin-induced plasma extravasation is mediated solely through NK-1 (SP-P) receptors or through multiple tachykinin receptors specific for individual tachykinins, we induced tachyphylaxis of the vascular responses by intradermal preinjection of each of the three mammalian tachykinins and examined the specificity of the tachyphylaxis. First, one of the tachykinins (50 I.il) or PBS (50 ul) was injected intradermally. Thirty minutes after the first injection, another injection of the same or a different tachykinin (50 ~1) was performed at the same injection site. Evans blue was administered 5 min before the second injection. No Evans blue extravasation was observed when the dye was administered more than 20 min after the first injection of tachykinins. The specificity of the tachyphylaxis was determined by using equipotent concentrations of tachykinins (3 x 10-7 M SP, 10-5 M NKB, and 2 x 10-5 M NKA). 4.
Drugs used
Substance P and Evans blue were purchased from Sigma Chemical Co. (St. Louis, MO). Neurokinin A, neurokinin B, physalaemin, eledoisin, kassinin, SPB-11, and SPl-g were purchased from Peninsula Laboratories (Belmont, CA).
Vol.
44, No.
16,
1989
Tachykinin
and Vascular
Permeability
1091
All of the tachykinins with the exception of NKB (2 x 10-3 M) and SP fragments were dissolved in 0.1 N acetic acid and then diluted with PBS. Neurokinin B (2 x 10-2 M) was dissolved in 10 N acetic acid and then diluted with PBS. Evans blue was dissolved in physiological saline.
5.
Data analysis
Data are reported as means l SD. statistical comparison of the data.
The paired Student's test was used for
Results 1.
Potency and rank order of mannnalian and nonmamnalian tachykinins for inducing plasma extravasation in guinea pig skin
Mammalian and nonmarnmalian tachykinins (10-g to 10-S M) induced plasma extravasation in guinea pig skin concentration-dependently when administered intradermally (fig. 1). Among mammalian tachykinins, the rank order of potency for inducing plasma extravasation in guinea pig skin was SP>NKB?.NKA (fig. 1, upper). The difference in the potency between SP and NKB (or NKA) was about a half log. Among nonmammalian tachykinins, the responses were equipotent (physalaemin?eledoisin=kassinin) (fig. 1, lower). These findings syggest that the tachykinin-induced plasma extravasation in guinea pig skin is an NK-1 (SPP)-type response [61.
SP6-11 (10-6 to 10-S M) induced plasma extravasation concentrationdependently (fig. 1, lower), and the potency was one log lower than SP. SPI-g (up to 10-5 M) did not induce plasma extravasation. These findings suggest that C-terminal hexapeptide of SP is important in increasing vascular permeability. 2.
Mammalian tachykinin-induced tachyphylaxis of the responses
Substance P, NKB, and NKA induced tachyphylaxis of the vascular responses concentration-dependently. Intradermal preinjection of SP, NKB, or NKA (10-8 to 10-5 M) decreased plasma extravasation induced by the second injection of the same tachykinin (10-5 M) depending on the concentration of tachykinin preinjected (fig. 2, upper). The maximum degree of tachyphylaxis that was produced, that is, 35% to 38% reduction in the responses induced by preinjections of 10-8 M tachykinins, correspond to approximately 90% inhibition of the responses from the concentration-response curves (fig. 1, upper). Tachyphylaxis of the vascular response induced by mansnalian tachykinins was tachykinin-specific. In SP tachyphylaxis (where SP was preinjected intradermally), only the response to SP was decreased, but the responses to NKB or NKA were the same as controls (preinjection of PBS) (fig. 2, lower). In NKB tachyphylaxis and NKA tachyphylaxis, only the responses to NKB and NKA, respectively, were decreased, but the responses to SP or NKA, and the responses to SP or NKB, were the same as controls, respectively. The results suggest that SP induced the response solely through NK-1 (SP-P) receptors, NKA solely through NK-2 (NKA) receptors, and NKB solely through NK-3 (NKB) receptors.
1092
Tachykinin and Vascular Permeability
Vol. 44, No. 16, 1989
Evans blue extravasation
80604020-
80 60
-9
-8 -7 Tachykinins
FIG.
-6 (log M)
-5
1.
mammalian (upper) and curves of Concentration-response nonmammalian (lower) tachykinin-induced plasma extravasation in guinea pig skin. Evans blue extravasation was measured 20 min after intradermal injection of 50 cjlof a tachykinin. 0 = SP, I = NKB, and A = NKA in the upper panel. 0 = physalaemin, D = eledosin, A = kassinin, 0 = SPg_11, and V = SPl-g in the lower panel. Data are means + SD for six experiments in the upper*panel and for three P< 0.025, significantly experiments in the lower panel. *PC 0.05, different from the mean of the response induced by NKB or NKA.
Tachykinin and Vascular Permeability
Vol. 44, No. 16, 1989
1093
Evans blue extravasation
-6 -7 -6
-6 -7 -6 SP
(log
SP
M)
NKB
(log
NKB
-6 -7 -6 M)
NKA
(log
M)
NKA
FIG. 2. Concentration-dependency (upper) and specificity (lower) of tachyphylaxis of mammalian tachykinin-induced plasma extravasation in guinea pig skin. Thirty minutes after the first intradermal injection of a tachykinin, the second injection was performed at the original injection site. Five minutes before the second injection, Evans blue was administered intravenously. In the upper panel, the concentrations of tachykinins used in the first injections were 10-6 to 10-6 M, and the second injection was made with the same tachykinin (10-6 M). In the lower panel, the equipotent concentrations of tachykinins used were the same in the first and second injections (3 x 10-T M SP, 10-6 M NK6, and 2 x 104 M NKA). The axis is normalized to the values that were obtained when the first injection was made with PBS (control). Oata are means * SO for four experiments in the upper panel and five experiments in the lower panel. lP c 0.025, **P c 0.005, significantly different from the mean of the control.
1094
Tachykinin and Vascular Permeability
vol. 44, No. 16, 1989
Discussion
In this study, we show that three mammlian tachykinins, SP, NKA, and NKB, increase vascular permeability in guinea pig skin through three different subtypes of tachykinin receptors, i.e., NK-1 (SP-P) receptors, NK-2 (NKA) receptors, and NK-3 (NKB) receptors, and that each tachykinin preferentially stimulates its specific receptors in the vascular response, i.e., SP, NKA, and NKB stimulate NK-1, NK-2, and NK-3 receptors, respectively. On the basis of the rank order of potency of mammalian and nonmammalian tachykinins for inducing plasma extravasation in guinea pig skin, we found that the vascular response was NK-l-type (fig. 1) [61. The vascular response was not inhibited by a histamine HI antagonist and seemed to be direct effects of tachykinins [lo]. We also found that the vascular response was C-terminal hexapeptidedependent (fig. 1). Then, to determine whether the apparent NK-l-type response is mediated solely through NK-1 receptors or through multiple tachykinin receptors, we induced tachyphylaxis of the response to each of the three mammalian tachykinins and determined the specificity. We found that the tachphylaxis was specific for each tachykinin and did not affect the responses to the other two tachykinins (fig. 2). Thus, the apparent NK-1 (SP-P)-type response of plasma extravasation in the skin is mediated through three different subtypes of tachykinin receptors. We interpret the fact that the responses induced by the three mammalian tachykinins are nearly equal as suggesting that the responses are mediated via three different tachykinin receptors. If the responses were mediated solely through NK-1 (SP-P) receptors, the responses to NKA and NKB should have been much less than the response to SP, because NK-1 receptors have tens to hundreds of times less For the same reason Clll, if the response to SP affinity for NKA and NKB [91. was mediated mainly through NK-2 (NKA) receptors, the response to SP should have been hundreds of times less than the response to NKA. Because plasma extravasation is caused mainly by contraction of endothelial cells of capillaries and venules [121, our findings can be interpreted in terms of three different tachykinin receptors located in endothelial cells of cutaneous blood vessels. Indeed, an autoradiographic study showed SP binding sites in the endothelium of the blood vessels [131. Our findings are in close agreement with the finding that the potency of NKB and NKA for releasing arterial relaxing factor(s) from the endothelium of rabbits was one-fourth to one-fifth of that of SP [141. Thus, the possible existence of three different tachykinin receptors in the endothelium of cutaneous blood vessels suggests that all three endogenous tachykinins may play a physiological role in increasing vascular permeability in the skin. In this regard, it is interesting that SP and NKA are co-localized in certain primary afferent neurones, whereas NKB is localized in interneurones or ascending pathways rather than in primary afferents [151. Therefore, we speculate that each tachykinin may be released to increase vascular permeability from different neurones, probably by different stimuli. Finally, the precise mechanism of the tachyphylaxis in tachykinin-induced plasma extravasation is unknown. However, the tachyphylaxis is a homologoustype desensitization, not a heterologous one, so tachykinin receptors themselves are most likely involved in the phenomenon, and phosphorylation and/or internalization of the receptors might be responsible for inducing the unresponsiveness of the receptor-mediated pathways as indicated in other experimental systems [161. In summary, we have shown that SP, NKA, and NKB induce plasma extravasation in guinea pig skin specifically through NK-1, NK-2, and NK-3 receptors, respectively.
Vol. 44, No. 16, 1989
Tachykinin and Vascular Permeability
1095
Acknowledgments We thank Ms. I. Ueki for technical assistance and Ms. B. Cost and Ms. P. Snell for assistance in preparing the manuscript. This study was supported in part by NIH Program Project Grant HL-24136. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
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