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Substance P induced hydrolysis of inositol phospholipids in rat skin in an in vivo model of inflammation
Neuropeprides (1989) 13,191-196 @ Longman Group UK Ltd 1989
Substance P Induced Hydrolysis of lnositol Phospholipids in Rat Skin in an In Vivo Model of Inflammation K. L. THOMAS,
P. V. ANDREWS,
Z. KHALIL and R. D. HELME
National Research institute of Gerontology 3052, Australia (reprint request to R.D.H.I.
and Geriatric Medicine,
Mount Royal Hospital, Parkville,
Abstract-The present study was undertaken to study the ability of substance P (SP) to induce inositol phospholipid (IP) hydrolysis measured as inositol mono-phosphate (IPl) accumulation, in an in viva blister model of neurogenic inflammation in the rat hind footpad. SP was found to induce IPl accumulation in a concentration dependent manner. The use of SP analogues (SP5_,, and SP1_7) indicated that the response is mainly mediated by the C-terminal sequence of the peptide. The response was significantly reduced by the SP antagonist spantide, suggesting that the response is mostly due to activation of the SP receptor on small diameter vessels. Capsaicin pretreatment did not have an effect on the ability of SP to induce the response. Experiments with mepyramine suggest that the response is also partly mediated by SP induced histamine release from mast cells. This is the first study to provide direct evidence for phosphoinositide mediated SP effects in the skin.
introduction
Neurogenic inflammation in the form of vasodilatation and plasma extravasation (PE) is known to be mediated by peptides released from small diameter sensory fibres. Evidence has been accumulated to suggest that SP is the proposed mediator (1). Previous studies in our laboratory using a blister model in the rat hind footpad have shown that perfusion of SP over the blister base induces PE and vasodilatation responses (2). In addition,
Date received 23 November 1988 Date accepted 28 November 1988
we have provided evidence that PE is mediated by activation of the NK-1 receptor (3) on post capillary venules (4). The receptor mechanism underlying this activation is unknown. There has been no conclusive evidence to link cyclic AMP metabolism with activation of SP receptors but there is increasing evidence linking SP mediated effects to the hydrolysis of inositol phospholipids. SP has been shown to induce IP hydrolysis in a variety of tissues including areas of the brain such as the hypothalamus (5). Other studies have shown SP to be active in tissues such as guinea-pig ileum (5, 6), parotid gland (7), adrenal gland (8), urinary bladder (9) 191
192
and rabbit iris sphincter muscle (10). There have not been any studies investigating IP linked activation by SP in the skin. There have been few studies undertaken in vivo. These have investigated IP hydrolysis in brain (11, 12) or rabbit iris smooth muscle (3). In the present study, we have investigated the possibility that SP activates a receptor to induce PE via IP hydrolysis. The direct effect of SP on small diameter vessels was tested using the SP antagonist spantide and the C- and N-terminal analogues of the SP peptide. In addition, the involvement of primary afferent sensory fibres and mast cells in the response to SP was investigated using neonatal capsaicin pretreatment and mepyramine respectively.
Materials and Methods Outbred Sprague-Dawley rats were obtained from Monash University Central Animal House. The weight of the rats ranged between 250-3008. Anaesthesia was induced with pentobarbitone sodium (Nembutal 6Omg/ml, Ceva Chem. Aust. Pty. Ltd.) 65mglkg i.p. Venous access was obtained with an indwelling catheter in the saphenous vein of the left leg. Anaesthesia was maintained with additional Nembutal as required. All animals were given a fluid load of normal saline 1 ml/50g i.v.. Body temperature was maintained at 37°C. Animals were killed at the end of each experiment by cervical dislocation. A blister was induced on the right hind footpad using a modification of a previously published method (14). A suction pressure of -40kPa was applied for 30 minutes using a metal suction cap heated to 40°C with a heating element. When a blister had been established, the surface epithelium was removed and the lower part of a perspex pefusion chamber was placed around the blister base. 2O@i of myo-[23H] inositol (Amersham TRK 807) was added to the blister base for 1 hour. On completion of the incubation period the remaining incubation fluid was removed and the top half of the perfusion chamber was placed over the blister base. The blister base was washed with Ringer’s solution containing 5 mM Inositol for one hour at a perfusion rate of 9.9mUhr (LKB Model
NEUROPEPTIDES
2232 Microperpex peristaltic pump.) to remove excess unincorporated myo-[23H] inositol, followed by perfusion for 10 minutes with Ringer’s containing 12mM lithium chloride. The blister base was perfused at 4mYhr with either agonist made up in Ringer’s solution containing 12mM lithium chloride or with Ringer’s with added lithium chloride. Immediately after the stimulation period the rat was killed and the blister base area including the underlying tissue was removed and frozen onto dry-ice. The tissue was homogenized in 10% TCA (Polytron homogenizer), left for 15 minutes on ice and centrifuged at 3000rpm for 15 minutes. The supernatant was removed and washed four times with 2ml diethyl ether, neutralised with an appropriate amount of 0.1 M NaOH and diluted to 10 ml with distilled water before being added to an anion exchange column. The water soluble inositol phosphates were separated using an anion exchange resin (BIORAD AG 1 x 8 100-200 mesh formate form.). The resin was stored as a 1:l slurry with 0.1 M Formic Acid. 1.5 mls of the slurry was pipetted into 10ml polypropylene columns (BIO-RAD Econocolumn Chromatography Columns) and the column was washed with 3 X 10m15 mM inositol to wash out any free label. Inositol mono-phosphate was eluted with 10ml of 0.2M ammonium forrnate made up in 0.1 M formic acid. Duplicate 1 ml aliquots were pipetted into 7ml scintillation vials (Canberra Packard) and 5mls of Instagel (Canberra Packard) added. Each vial was mixed thorougly and left for 24 hrs in the dark to reduce chemiluminescence. Samples were counted for 5 minutes each. Neonatal rats were pretreated at day 2-5 of life with capsaicin (Fluka) at a dose of 50mg/kg sub-cutaneously. The effectiveness of this treatment was tested on the day of experimentation using the eye-wipe test (15). Mepyramine (Sigma) was administered intraperitoneally at a dose of lOmg/kg, 1 hour before agonist perfusion. SP and spantide were obtained from Auspep. Histamine was obtained as a lmg/ml solution from David Bull Laboratories. All other chemicals were of reagent grade quality. All data is presented as mean rf: SEM. Statistical analysis was performed using one way analysis of
SUBSTANCE P INDUCED HYDROLYSIS
OF INOSITOL PHOSPHOLIPIDS
193
IN RAT SKIN
variance with differences between groups determined using the Duncan multiple range test at a significance level of P < 0.05.
1
Results In each figure the data is represented as % of the control value (387 + 14dpm). Incubation of the blister base with SP led to an accumulation of IPl in a concentration dependent manner (from 10e9M to lo-‘M) with an agonist contact time of 5 minutes (Fig 1). There was no significant accumulation of inositol bisphosphate (IP2) or inositol trisphosphate (IP3) (data not shown). Substance P at a concentration of lo-‘M gave a response of 203% of control and was subsequently used in all further experiments. SP5_rl, a peptide with a C-terminal common to SP, was tested for its effect on PI hydrolysis. To facilitate comparison to the SP response, a concentration of lo-‘M was used. The results show that SPs.rr induced a response which represents 45% of the response to SP (Fig 2). On the other hand, when SP1_,, a peptide with an N-terminal sequence common to SP, was perfused at a concentration of 10e5M, no increase above control was detected. Pretreatment with capsaicin had no effect on the SP induced accumulation of IPl (Fig 3). When the spantide at 10m5M was perfused by itself for 5 minutes there was no increase in IPl above control levels. This concentration of spantide was used because it has been found to be optimal in our system (16). When perfused in conjunction with SP, spantide significantly reduced the response to SP from 788 dpm to 423 dpm a reduction of 90% (Fig 3). When mepyramine (an Hr antagonist) was injected at lOmg/kg, 1 hour prior to agonist stimulation, it resulted in a significant decrease in the response to SP from 788dpm to 578dpm, a reduction of 27%. Histamine was then used to test its effect on IP hydrolysis. It was first used at 1 ~J,M, where there was no increase above control. At lmM, Histamine induced an increase of 31% above control (Fig 4). Analysis of variance between SP lo-‘M and the treatment groups gave an F value (F4, 38 = 13.30) with a significance of P < 0.0001. Further analysis using the Duncan multiple range test revealed
_. s?
i
lo-‘Y
SP lo-%4
sf
lo-%
Fig 1 The effect of SP on IPl accumulation at various concentrations. Columns are the mean + SEM. The number of animals in each group are: 10m9M, n = 6; lo-‘M. n = 6; lo-‘M, n = 11.
1
I
.
h
L
I
:-TERM
.
N-TERM
L
I
L
SP 10-h
1
.-
Fig 2 The effect of C- and N-terminals analogues of SP on IPl accumulation. Columns are the mean + SEM. The number of animals in each group are: C-terminal, n = 6; N-terminal, n = 6; lO--‘M, n = 11.
d E 8
150 l
8 W.
7
50
I:I sPANno
sP+&wr SPlirsy
SPpi
Fig 3 The IPl accumulation induced by SP 10e5 M in animals pretreated with capsaicin or spantide (lo-‘M). Columns are the mean + SEM. The number of animals in each group are: spantide alone, n = 4; SP + spantide, n = 12; SP lo-‘M alone, II = 11; SP + capsaicin, n = 6. The * represents significant difference from SP 10msM alone.
194
NEUROPEPTIDES
htw +
SP
w
lo-Ju
puwsr
myMsI
Fig 4 The effect of mepyramine on the SP lo-‘M induced accumulation of IPq and the IPl accumulation induced by histamine. Columns are the mean + SEM. The number of animals in each group are: mepyramine + SP, n = 6; SP 10L5M alone, n = 11; PM histamine, n = 6; mM histamine, n = 6. The * represents a significant difference from the SP lo-‘M alone group.
significant differences at the 0.05 level between SP alone and the SP + spantide and the SP + mepyramine groups.
Discussion The role of SP in neurogenic inflammation has been extensively studied in our laboratory using an in vivo blister model in rat skin. We have shown that SP is capable of inducing PE and vasodilatation responses via activation of the NK-1 receptor, through its C-terminal end (3). This tachykinin receptor is thought to be located on post capillary venules and arterioles. To further investigate the mechanism underlying the PE response to SP, we have investigated the possibility that post receptor mechanisms may be mediated by IP hydrolysis. This mechanism of receptor activation has been shown to be mediated by SP in other systems (5-10). The effect of SP on the hydrolysis of phosphoinositides was studied in the presence of lithium which inhibits the enzyme myo-inositol-l-phosphate and thus amplifies the accumulation of IPl. As shown in Figure 1, incubation of the blister base with SP resulted in accumulation of IPl in a concentration dependant manner approaching
maximal at 10e5M. This concentration of SP was shown to be effective in other systems (5, 17). In our system however, there was no detectable increase in IP2 and IP3 (data not shown). A similar increase in IPl and no reported increase in IP2 or IP3 was also shown in guinea pig ileum and rat hypothalamus (5). The potency of SP in causing IPl accumulation and the percentage increases above control are comparable to other studies (6, 18). It has been suggested that the PE response is a function of the C-terminal sequence of SP, and in this study 45% of the response to SP appeared to be mediated by its C-terminal. The N-terminal sequence of SP appears to have a negligible role in the response to SP. This does not exclude the possibility that the N-terminal sequence may activate SP receptors by mechanisms other than IP hydrolysis. The PE response is a function of small diameter sensory fibres. Incubation of the blister base with SP could indirectly activate sensory nerve terminals to release other mediators which in turn induce IP hydrolysis. To exclude the possibility that sensory fibres were involved in this response, the same experiments were performed in capsaicin pretreated rats. Neonatal capsaicin treatment is known to cause degeneration of primary afferent neurons (19). Incubation of the blister base with SP in these animals induced an accumulation of IPl similar to that obtained in control rats. This suggests that the accumulation if IPl is due to the action of SP applied to the blister base rather than a function of other mediators released from sensory nerves or by activation of preterminal receptors. The SP antagonist spantide, when perfused in conjunction with SP, significantly decreased the response to SP by 90%. This is further evidence suggesting that the IPl accumulation effect is mainly due to a C-terminal receptor activation by SP. Histamine has been implicated in the PE response to SP (20) and evidence has been provided that SP induced PE is partly mediated via histimine release from mast cells (16). In order to test the involvement of histamine in IPl accumulation, rats were treated with the Hi antagonist mepyramine. The results in figure 4, using SP
SUBSTANCE P INDUCED HYDROLYSIS OF INOSITOL PHOSPHOLIPIDS IN RAT SKIN
incubation in mepyramine treated rats suggest that endogenous histamine could be involved in the response to SP via activation of the Hi receptor. It has previously been shown that histamine induced PE at mM concentration (21) and it was observed in this study that IPl accumulation occurred at this concentration. In conclusion, the present results provide evidence for the first time in vivo to suggest that activation of SP receptors in the rat skin is mediated via IP hydrolysis. The response obtained was independent of capsaicin sensory fibres and was mainly due to direct activation of SP receptors on post capillary venules. The use of SP analogues indicated that this activation is mainly a function of the C-terminal sequence of SP. In addition the response maybe partly mediated via endogenous histamine release from mast cells activating H1 receptors. The present study has implicated IP hydrolysis as one of the underlying receptor activation mechanisms by which neurogenic inflammation is induced in rat skin.
Acknowledgements This project was supported by a grant from the National Health and Medical Research Council of Australia.
References Lembeck, F. and Holzer, P. (1979). Substance P as neurogenic mediator of antidromic vasodilatation and neurogenic plasma extravasation. Naunyn Schmiedeberg’s Archives of Pharmacolgy 310: 175-183. Andrews, P. V. and Helme, R. D. (1987). Tachykinin induced plasma extravasation is mediated through the SP-P receptor. In: Henry, J. L., Couture, R., Cuello, A. C., Pelletier, G., Quirion, R. and Regoli, D. (eds.) Substance P and Neurokinins. Springer Verlag. New York, ~203-204. Andrews, P. V., Thomas, K. L. and Helme, R. D. Neurogenic plasma extravasation in rat skin is mediated by the NK-1 receptor. Manuscript submitted. Kenins, P., Hurley, J. V. and Bell, C. (1984). The role of substance Pin the axon reflex in the rat. British Journal of Dermatology 111: 551-559. Watson, S. P. and Downes, C. P. (1983). Substance P induced hydrolysis of inositol phospholipids in guinea-pig ileum and rat hypothalamus. European Journal of Pharmacology 93: 245-253.
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6. Holzer, P. and Lippe, I. Th. (1985). Substance P action on phosphoinositides in guinea-pig intestinal muscle: A possible transduction mechanism? Naunyn Schmiedeberg’s Archive of Pharmacology 329: 50-55. 7. Berridge, M. J., Downes, C. P. and Hanley, H. R. (1982). Lithium amplifies agonist dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206: 587-595. 8. Minenko, A. and Oehme, P. (1987). Substance P action on inositol phospholipids in rat adrenal medulla slices. Biomed. Biochim. Acta 46: 461-467. 9. Bristow. D. R., Curtis, N. R., Suman-Chauhan, N.. Watling. K. J. and Williams, B. J. (1987). Effects of tachykinins on inositol phospholipid hydrolysis in slices of hamster urinary bladder. British Journal of Pharmacology 90: 211-217. 10. Yousufzai. S. Y. K., Akhtar, R. A. and Abdel-Latif, A. A. (1986). Effects of substance P on inositol triphosphate accumulation, on contractile responses and on arachidonic acid release and prostaglandin synthesis in rabbit iris sphincter muscle. Experimental Eye Research 43: 215-226. 11. Soukup, J. and Schanberg, S. (1982). Involvement of alpha noradrenergic receptors in mediation of brain polyphosphoinositide metabolism in vivo. Journal of Pharmacology and Experimental Therapeutics 222: 209-214. 12. Pickard. M. R. and Hawthorne, J. N. (1978). The labelling of nerve ending phospholipids in guinea-pig brain in viva and the effect of electrical stimulation on phosphatidylinositol metabolism in prelabelled synaptosomes. Journal of Neurochemistry 30: 145-155. 13. Abdel-Latif, A. A., Green, K., Smith, J. P., McPherson Jr.. J. C. and Matheny. J. L. (1978). Norephinephrinestimulated breakdown of triphosphoinositide of rabbit iris smooth muscle: Effects of surgical sympathetic denervation and in vivo electrical stimulation of the sympathetic nerve of the eye. Journal of Neurochemistry 30: 517-525. 14 Helme, R. D., White, D. M. and Andrews. P. V. (198.5). Neurogenic inflammation in skin blisters. Experimental Brain Research 59: 382-387. 15. Helme. R. D.. Eglezos. A., Dandie, G. W.. Andrews, P. V. and Boyd. R. L. (1987). The effect of substance P on the regional lymph node antibody response to antigenic stimulation in capsaicin pretreated rats. Journal of Immunology 139: 3470-3473. 16 Khali. Z. and Helme, R. D. Sequence of events in substance P medicated plasma extravasation in rat skin. Manuscript submitted. 17. Watson. S. P. (1984). The action of substance P on contraction, inositol phospholipids and adenylate cyclase in rat small intestine. Biochem. Pharmacol. 33: 3733-3737. 18. Bailey, S. J., Lippe. I.Th. and Holzer. P. (1987). Effect of the tachykinin antagonist [D-pro’. D-trp’~“~“‘] Substance P-(4-1 1). on tachykinin- and histamine-induced inositol phosphate generation in intestinal smooth muscle. Naunyn Schmeideberg’s Archives of Pharmacology 335: 296-300. 19. Jancso. N.. Jancso-Gabor, A. and Szolcsanyi, J. (1967). Direct evidence for neurogenic inflammation and its pre-
196 vention by denervation by pretreatment with capsaicin. British Journal of Pharmacology 31: 138-151. 20. Foreman, J. C., Jordan, C. C., Oehme, P. and Renner, H. (1983). Structure-activity relationships for some substance P-related peptides that cause wheal and flare reactions in human skin. Journal of Physiology 335: 449-465.
NEUROPEPTIDES 21. Woodward, D. F., Owen, D. A. A., Pipkin, H. A. and Ledgard, S. E. (1985). Histamine involvement in the local and systematic microvascular effects produced by intraderma1 substance P. Agents and Actions 17: 126-130.
Substance P Induced Hydrolysis of lnositol Phospholipids in Rat Skin in an In Vivo Model of Inflammation K. L. THOMAS,
P. V. ANDREWS,
Z. KHALIL and R. D. HELME
National Research institute of Gerontology 3052, Australia (reprint request to R.D.H.I.
and Geriatric Medicine,
Mount Royal Hospital, Parkville,
Abstract-The present study was undertaken to study the ability of substance P (SP) to induce inositol phospholipid (IP) hydrolysis measured as inositol mono-phosphate (IPl) accumulation, in an in viva blister model of neurogenic inflammation in the rat hind footpad. SP was found to induce IPl accumulation in a concentration dependent manner. The use of SP analogues (SP5_,, and SP1_7) indicated that the response is mainly mediated by the C-terminal sequence of the peptide. The response was significantly reduced by the SP antagonist spantide, suggesting that the response is mostly due to activation of the SP receptor on small diameter vessels. Capsaicin pretreatment did not have an effect on the ability of SP to induce the response. Experiments with mepyramine suggest that the response is also partly mediated by SP induced histamine release from mast cells. This is the first study to provide direct evidence for phosphoinositide mediated SP effects in the skin.
introduction
Neurogenic inflammation in the form of vasodilatation and plasma extravasation (PE) is known to be mediated by peptides released from small diameter sensory fibres. Evidence has been accumulated to suggest that SP is the proposed mediator (1). Previous studies in our laboratory using a blister model in the rat hind footpad have shown that perfusion of SP over the blister base induces PE and vasodilatation responses (2). In addition,
Date received 23 November 1988 Date accepted 28 November 1988
we have provided evidence that PE is mediated by activation of the NK-1 receptor (3) on post capillary venules (4). The receptor mechanism underlying this activation is unknown. There has been no conclusive evidence to link cyclic AMP metabolism with activation of SP receptors but there is increasing evidence linking SP mediated effects to the hydrolysis of inositol phospholipids. SP has been shown to induce IP hydrolysis in a variety of tissues including areas of the brain such as the hypothalamus (5). Other studies have shown SP to be active in tissues such as guinea-pig ileum (5, 6), parotid gland (7), adrenal gland (8), urinary bladder (9) 191
192
and rabbit iris sphincter muscle (10). There have not been any studies investigating IP linked activation by SP in the skin. There have been few studies undertaken in vivo. These have investigated IP hydrolysis in brain (11, 12) or rabbit iris smooth muscle (3). In the present study, we have investigated the possibility that SP activates a receptor to induce PE via IP hydrolysis. The direct effect of SP on small diameter vessels was tested using the SP antagonist spantide and the C- and N-terminal analogues of the SP peptide. In addition, the involvement of primary afferent sensory fibres and mast cells in the response to SP was investigated using neonatal capsaicin pretreatment and mepyramine respectively.
Materials and Methods Outbred Sprague-Dawley rats were obtained from Monash University Central Animal House. The weight of the rats ranged between 250-3008. Anaesthesia was induced with pentobarbitone sodium (Nembutal 6Omg/ml, Ceva Chem. Aust. Pty. Ltd.) 65mglkg i.p. Venous access was obtained with an indwelling catheter in the saphenous vein of the left leg. Anaesthesia was maintained with additional Nembutal as required. All animals were given a fluid load of normal saline 1 ml/50g i.v.. Body temperature was maintained at 37°C. Animals were killed at the end of each experiment by cervical dislocation. A blister was induced on the right hind footpad using a modification of a previously published method (14). A suction pressure of -40kPa was applied for 30 minutes using a metal suction cap heated to 40°C with a heating element. When a blister had been established, the surface epithelium was removed and the lower part of a perspex pefusion chamber was placed around the blister base. 2O@i of myo-[23H] inositol (Amersham TRK 807) was added to the blister base for 1 hour. On completion of the incubation period the remaining incubation fluid was removed and the top half of the perfusion chamber was placed over the blister base. The blister base was washed with Ringer’s solution containing 5 mM Inositol for one hour at a perfusion rate of 9.9mUhr (LKB Model
NEUROPEPTIDES
2232 Microperpex peristaltic pump.) to remove excess unincorporated myo-[23H] inositol, followed by perfusion for 10 minutes with Ringer’s containing 12mM lithium chloride. The blister base was perfused at 4mYhr with either agonist made up in Ringer’s solution containing 12mM lithium chloride or with Ringer’s with added lithium chloride. Immediately after the stimulation period the rat was killed and the blister base area including the underlying tissue was removed and frozen onto dry-ice. The tissue was homogenized in 10% TCA (Polytron homogenizer), left for 15 minutes on ice and centrifuged at 3000rpm for 15 minutes. The supernatant was removed and washed four times with 2ml diethyl ether, neutralised with an appropriate amount of 0.1 M NaOH and diluted to 10 ml with distilled water before being added to an anion exchange column. The water soluble inositol phosphates were separated using an anion exchange resin (BIORAD AG 1 x 8 100-200 mesh formate form.). The resin was stored as a 1:l slurry with 0.1 M Formic Acid. 1.5 mls of the slurry was pipetted into 10ml polypropylene columns (BIO-RAD Econocolumn Chromatography Columns) and the column was washed with 3 X 10m15 mM inositol to wash out any free label. Inositol mono-phosphate was eluted with 10ml of 0.2M ammonium forrnate made up in 0.1 M formic acid. Duplicate 1 ml aliquots were pipetted into 7ml scintillation vials (Canberra Packard) and 5mls of Instagel (Canberra Packard) added. Each vial was mixed thorougly and left for 24 hrs in the dark to reduce chemiluminescence. Samples were counted for 5 minutes each. Neonatal rats were pretreated at day 2-5 of life with capsaicin (Fluka) at a dose of 50mg/kg sub-cutaneously. The effectiveness of this treatment was tested on the day of experimentation using the eye-wipe test (15). Mepyramine (Sigma) was administered intraperitoneally at a dose of lOmg/kg, 1 hour before agonist perfusion. SP and spantide were obtained from Auspep. Histamine was obtained as a lmg/ml solution from David Bull Laboratories. All other chemicals were of reagent grade quality. All data is presented as mean rf: SEM. Statistical analysis was performed using one way analysis of
SUBSTANCE P INDUCED HYDROLYSIS
OF INOSITOL PHOSPHOLIPIDS
193
IN RAT SKIN
variance with differences between groups determined using the Duncan multiple range test at a significance level of P < 0.05.
1
Results In each figure the data is represented as % of the control value (387 + 14dpm). Incubation of the blister base with SP led to an accumulation of IPl in a concentration dependent manner (from 10e9M to lo-‘M) with an agonist contact time of 5 minutes (Fig 1). There was no significant accumulation of inositol bisphosphate (IP2) or inositol trisphosphate (IP3) (data not shown). Substance P at a concentration of lo-‘M gave a response of 203% of control and was subsequently used in all further experiments. SP5_rl, a peptide with a C-terminal common to SP, was tested for its effect on PI hydrolysis. To facilitate comparison to the SP response, a concentration of lo-‘M was used. The results show that SPs.rr induced a response which represents 45% of the response to SP (Fig 2). On the other hand, when SP1_,, a peptide with an N-terminal sequence common to SP, was perfused at a concentration of 10e5M, no increase above control was detected. Pretreatment with capsaicin had no effect on the SP induced accumulation of IPl (Fig 3). When the spantide at 10m5M was perfused by itself for 5 minutes there was no increase in IPl above control levels. This concentration of spantide was used because it has been found to be optimal in our system (16). When perfused in conjunction with SP, spantide significantly reduced the response to SP from 788 dpm to 423 dpm a reduction of 90% (Fig 3). When mepyramine (an Hr antagonist) was injected at lOmg/kg, 1 hour prior to agonist stimulation, it resulted in a significant decrease in the response to SP from 788dpm to 578dpm, a reduction of 27%. Histamine was then used to test its effect on IP hydrolysis. It was first used at 1 ~J,M, where there was no increase above control. At lmM, Histamine induced an increase of 31% above control (Fig 4). Analysis of variance between SP lo-‘M and the treatment groups gave an F value (F4, 38 = 13.30) with a significance of P < 0.0001. Further analysis using the Duncan multiple range test revealed
_. s?
i
lo-‘Y
SP lo-%4
sf
lo-%
Fig 1 The effect of SP on IPl accumulation at various concentrations. Columns are the mean + SEM. The number of animals in each group are: 10m9M, n = 6; lo-‘M. n = 6; lo-‘M, n = 11.
1
I
.
h
L
I
:-TERM
.
N-TERM
L
I
L
SP 10-h
1
.-
Fig 2 The effect of C- and N-terminals analogues of SP on IPl accumulation. Columns are the mean + SEM. The number of animals in each group are: C-terminal, n = 6; N-terminal, n = 6; lO--‘M, n = 11.
d E 8
150 l
8 W.
7
50
I:I sPANno
sP+&wr SPlirsy
SPpi
Fig 3 The IPl accumulation induced by SP 10e5 M in animals pretreated with capsaicin or spantide (lo-‘M). Columns are the mean + SEM. The number of animals in each group are: spantide alone, n = 4; SP + spantide, n = 12; SP lo-‘M alone, II = 11; SP + capsaicin, n = 6. The * represents significant difference from SP 10msM alone.
194
NEUROPEPTIDES
htw +
SP
w
lo-Ju
puwsr
myMsI
Fig 4 The effect of mepyramine on the SP lo-‘M induced accumulation of IPq and the IPl accumulation induced by histamine. Columns are the mean + SEM. The number of animals in each group are: mepyramine + SP, n = 6; SP 10L5M alone, n = 11; PM histamine, n = 6; mM histamine, n = 6. The * represents a significant difference from the SP lo-‘M alone group.
significant differences at the 0.05 level between SP alone and the SP + spantide and the SP + mepyramine groups.
Discussion The role of SP in neurogenic inflammation has been extensively studied in our laboratory using an in vivo blister model in rat skin. We have shown that SP is capable of inducing PE and vasodilatation responses via activation of the NK-1 receptor, through its C-terminal end (3). This tachykinin receptor is thought to be located on post capillary venules and arterioles. To further investigate the mechanism underlying the PE response to SP, we have investigated the possibility that post receptor mechanisms may be mediated by IP hydrolysis. This mechanism of receptor activation has been shown to be mediated by SP in other systems (5-10). The effect of SP on the hydrolysis of phosphoinositides was studied in the presence of lithium which inhibits the enzyme myo-inositol-l-phosphate and thus amplifies the accumulation of IPl. As shown in Figure 1, incubation of the blister base with SP resulted in accumulation of IPl in a concentration dependant manner approaching
maximal at 10e5M. This concentration of SP was shown to be effective in other systems (5, 17). In our system however, there was no detectable increase in IP2 and IP3 (data not shown). A similar increase in IPl and no reported increase in IP2 or IP3 was also shown in guinea pig ileum and rat hypothalamus (5). The potency of SP in causing IPl accumulation and the percentage increases above control are comparable to other studies (6, 18). It has been suggested that the PE response is a function of the C-terminal sequence of SP, and in this study 45% of the response to SP appeared to be mediated by its C-terminal. The N-terminal sequence of SP appears to have a negligible role in the response to SP. This does not exclude the possibility that the N-terminal sequence may activate SP receptors by mechanisms other than IP hydrolysis. The PE response is a function of small diameter sensory fibres. Incubation of the blister base with SP could indirectly activate sensory nerve terminals to release other mediators which in turn induce IP hydrolysis. To exclude the possibility that sensory fibres were involved in this response, the same experiments were performed in capsaicin pretreated rats. Neonatal capsaicin treatment is known to cause degeneration of primary afferent neurons (19). Incubation of the blister base with SP in these animals induced an accumulation of IPl similar to that obtained in control rats. This suggests that the accumulation if IPl is due to the action of SP applied to the blister base rather than a function of other mediators released from sensory nerves or by activation of preterminal receptors. The SP antagonist spantide, when perfused in conjunction with SP, significantly decreased the response to SP by 90%. This is further evidence suggesting that the IPl accumulation effect is mainly due to a C-terminal receptor activation by SP. Histamine has been implicated in the PE response to SP (20) and evidence has been provided that SP induced PE is partly mediated via histimine release from mast cells (16). In order to test the involvement of histamine in IPl accumulation, rats were treated with the Hi antagonist mepyramine. The results in figure 4, using SP
SUBSTANCE P INDUCED HYDROLYSIS OF INOSITOL PHOSPHOLIPIDS IN RAT SKIN
incubation in mepyramine treated rats suggest that endogenous histamine could be involved in the response to SP via activation of the Hi receptor. It has previously been shown that histamine induced PE at mM concentration (21) and it was observed in this study that IPl accumulation occurred at this concentration. In conclusion, the present results provide evidence for the first time in vivo to suggest that activation of SP receptors in the rat skin is mediated via IP hydrolysis. The response obtained was independent of capsaicin sensory fibres and was mainly due to direct activation of SP receptors on post capillary venules. The use of SP analogues indicated that this activation is mainly a function of the C-terminal sequence of SP. In addition the response maybe partly mediated via endogenous histamine release from mast cells activating H1 receptors. The present study has implicated IP hydrolysis as one of the underlying receptor activation mechanisms by which neurogenic inflammation is induced in rat skin.
Acknowledgements This project was supported by a grant from the National Health and Medical Research Council of Australia.
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