Neuropeptides (1992) 22, 137-141 0 Longman Group UK Ltd 1992
Substance P Augments the Rate of Vasodilation Induced by Calcitonin Gene-related Peptide in Porcine Ophthalmic Artery In Vitro M. B. VINCENT*, L. R. WHITE, T. ELSAS, G. QVIGSTAD and 0. SJAASTAD Department of Neurology, Trondheim University Hospital, Norway. *Department of Neurology, University Hospital, Federal University of Rio de Janeiro, 21949 Rio de Janeiro, Brazil (Reprint requests to MBV)
Abstract - Peptides may function as neurotransmitters liberated antidromically by sensory nerve fibres, provoking vascular responses having potential importance in some neurological disorders. Dose-response relaxation curves induced by substance P (SP) and calcitonin gene related peptide (CGRP) have been studied in porcine ophthalmic arteries in vitro. Both peptides induced vasodilation when tested separately (CGRP >> SP). Because of the putative interactions between such peptides in this vascular territory, a computerised system was also used for analysing over time the response to a single addition of either 10q M CGRP, lo4 M SP or a combination of 10q M SP + lOa M CGRP. SP did not augment the maximum relaxation induced by CGRP alone, but increased significantly the rate of relaxation during the initial phase of the response. The effect induced by the SP+CGRP combination was stronger than the sum of the individual SP and CGRP-induced relaxations during the first 4 min of the response, which suggests a SP-CGRP synergism in this artery.
Introduction Neuropeptides have been implicated in the pathophysiology of different neurological diseases (1). They also seem to be important in cerebral flow regulation (2) and have been suggested to be involved in headaches (3,4). Several neuropeptides may coexist with ‘classical’ neurotransmitters within the Date received 2 December 199 1 Date accepted 5 February 1992 Address for correspondence: Department of Neurology, Trondheim University Hospital, N-7006 Trondheim, Norway.
same fibres (5), possibly having intricate interactions not yet understood. The undecapeptide substance P (SP) is known to be present in trigeminal sensory fibres, as well as calcitonin gene-related peptide (CGRP), a 37 aminoacid peptide encoded by the calcitonin gene (6). SP-CGRP interactions have already been described in several studies. Intravitreal injections of CGRP in rabbits did not change the pupillar diameter, but the miosis produced by SP and CGRP in combination was greater than that produced by SP alone (7). The long lasting CGRP-induced vasodila137
138 tor response observed after an intradermal injection in humans was much shorter if SP was also injected, a phenomenon probably involving mast cell activation (8). CGRP potentiated the SP-induced biting and scratching behaviour when injected intrathetally in rats, whereas the administration of CGRP alone produced no behavioural changes (9). CGRP alone produced no release of excitatory aminoacids if infused into the rat dorsal spinal cord, but potentiated the apparent SP-induced release of taurine (10). CGRP was also shown to inhibit SP degradation by acting on an SP endopeptidase isolated from human CSF (11). The ocular and forehead circulation may play an important role in the pathophysiology of headaches. For instance, conjunctival injection, vasodilation, and lacrimation are known to occur in cluster headache (12). Both SP and CGRP are known to induce vasodilation in different arteries in the head (2), but the combined effects of SP and CGRP in this vascular bed, which may occur in vivo following sensory fibre activation, are unknown. The objective of this study was to investigate the vasodilation induced by CGRP and SP, separately and combined, in porcine ophthalmic artery in vitro.
Materials and Methods The entire orbital contents were removed from pigs anaesthetised with a-choralose and killed with a lethal dose of pentobarbital i.v., placed in iced, 02bubbled buffer solution (NaCl 119, KC1 4.6, CaCL 1.5, MgCh 1.2, NaHC03 10, NaH2P04 1.2, glucose 11. Values in 10” M) and kept at 4°C for 15 17 h. Retroocular segments of the ophthalmic artery were gently dissected with the aid of a stereomicroscope. Artery slices (1 to 3 mm) were mounted on two Lshaped steel holders (0.1 mm diameter), where one of the holders was connected to a Grass FT03C force-displacement transducer. For recording the arterial constriction forces, the transducers were linked to an Analog Digital Instruments MacLab Analog-Digital convertor through a Transbridge TBM4 preamplifier (World Precision Instruments, Inc, New Haven CT, USA). The digitalized data were analysed by MacLab’s Chart/4 software in an Apple Macintosh SE 1140 computer. The holders were immersed in 5 ml buffer solution (37’C, pH
NEUROPEPTIDES
7.4) which was continuously bubbled with a 5% C02, 95% O2 mixture. A passive tension of 4-5 mN was applied to the segments followed by an equilibration period of 1 - 1.5 h, until the tension was stable. The buffer solution was changed every 10 min throughout the equilibration period. The arteries were first contracted by 10-j M PGF2, and when a stable reaction was reached, the relaxation induced by the peptides was studied. PGF2, at a concentration of 10-j M was chosen as the best pre-contractor agent, since it proved to induce stable and long lasting contractions in this artery, suitable for performing dose-response experiments (data not shown). In order to obtain dose response relaxation curves, SP (acetate salt) or a-CGRP (human, synthetic) were added cumulatively in logarithmically increasing concentrations (SP: IO-loto 10” M. CGRP: lo-lo to lo-’ M). For studying the induced relaxation over 1 h, the time peptides were added was accurately recorded by the computer, and the relaxing response continuously registered throughout the following 60 min. SP experiments were terminated after 30 min since the SP-induced relaxations were short-lasting. The computer recorded the constriction forces every 6 s. Average curves representing the peptides activity over time were obtained from the digitalized values stored on the computer. The experiments were performed in parallel baths using consecutive slices of the same artery. A new animal was used for each set of experiments. The data are presented as the mean + S.E.M. Statistical analysis was carried out with Student’s t-test. Values of p < 0.05 were considered significant. All substances were added in 50 pl aliquots. Drugs were purchased from Sigma Chemical Co., St. Louis, MO, USA and were diluted in 0.9% NaCl containing 10” M ascorbic acid.
Results Dose-response experiments Figure 1 shows the relaxations obtained by adding cumulatively SP (1 O-loto 10” M) or CGRP (1 VI0 to 1V7 M) to isolated porcine ophthalmic arteries precontracted with 1V5 M PGFza. At lo-lo M, both peptides induced a relaxation of around 5%, while at 1O-9M, the CGRP-induced relaxation was higher
SP AUGMENTS
THE RATE OF CGRP IN PORCINE
OPHTHALMIC
ARTERY
139
IN VITRO
80 c .o iii
80
3 E
40
s
20
-10
-9
-8
-7
-8
-5
[peptide] (log M) Fig. 1 Relaxations induced by adding cumulatively, CGRP (lWO to lO_’M) or SP (lo-l0 to 1k5 M) to isolated porcine ophthalmic arteries pre-contracted by 1tYsM PGF*, in vitro. At 1W0 and lo+ M: no significant difference. At 10-s M and 1W M: p < 0.00001. Values represent the mean k S.E.M., n = 9.
than that induced by SP, but the difference was not statistically significant. However, at lo-* and lo-’ M, CGRP induced a relaxation significantly higher than SP (p < 0.00001). CGRP was not tested at concentrations higher than lo-’ M. SP was tested up to a concentration of 1O-5M, but induced its maximum effect at l@* M. Relaxation studies over time Figure 2a shows the relaxation curves induced by the addition of lo-* M CGRP, lo-8 M SP, or 1O-8M CGRP together with lo4 M SP, to porcine ophthalmic arteries pre-contracted by 1e5 M PGF2,. The reaction was studied continuously for approximately 1 h (CGRP) or 30 min (SP). The maximum relaxation induced by SP (around 15%), occurred on average after 3.0 + 0.6 min, and by CGRP (around 85%), after ca. 11.Of 0.7 min (p < 0.0000 1, Fig. 2a). When comparing the effects of CGRP alone and CGRP+SP, both curves were found to achieve maximum relaxation (around 85%) after about 10 min. Statistically, no difference was found in the values for the maximum effect (I,,), nor in the time taken to achieve I,,, (Table). Despite the tendency for a longer-lasting relaxation with the combination of peptides, no significant differences were found either in the time necessary to achieve a 50% recovery from the maximum effect (R,,), nor in the effects observed after 1 h (Table). In Figure 2b, these data are shown in greater detail to highlight the reaction during the first 7 min. A line
0
1
2
3
1
5
B
7
time (min)
Fig. 2 Average relaxation curves obtained by adding 1O-8M CGRP(n=8),1~8MP(n=7)and10-*MCGRP+10_8MSP (n = 7) to isolated porcine ophthalmic arteries pre-contracted by 1O-5M PGF2, in vitro. Fig. 2a: reaction durina 1h. S&es on the curves are electrical at-&acts and do not represent ;asoactivity. Fig. 2b: reaction during the first 7 mm. The dotted line represents the sum of the individual SP and CGRP reactions.
representing the sum of the SP effect plus the CGRP effect has also been included. This figure shows that the relaxation induced by the SP + CGRP combination was higher than the sum of each reaction individually during the first 4 min. The relaxation induced by SP declined after 3 min, whereas the CGRP and the CGRP + SP effects continued to increase. It is clear (Figs. 2b and 3) that there was an apparent difference in the initial rate of relaxation following peptide addition. Analysis of this early reaction (Fig. 3) showed that as early as 6 s after addition of peptides, the relaxation induced by SP together with CGRP was already stronger than that induced by CGRP alone, but the difference became significant (p < O.OS), first after 18 s. By 1 min the SP + CGRP combination induced a relaxation of 21.5 + 3.5%, with a corresponding relaxation of CGRP alone of only 4.7 f 1.4%. This difference was
140
NEUROPEPTIDES
Table
Time parameters SP+CGRP
CGRP
84.90+5.52 10.30 f 1.75 26.12 f 6.16 25.53 k 9.98
La, (%I tI,, (min) R0 (mm) E, ,, (%)
P
86.99f 5.20 11.02 f 0.75 24.47 f 4.2 1 20.59 f 5.76
NS NS NS NS
Maximum effect (I_, % relaxation), time necessary for reaching the maximum effect (tI,,,,, min), time necessary for a 50% recovery after maximum effect (&,, min) and effect present after 1 h (E, ,,, % relaxation) after adding 1W M CGRP and 10e8M CGRP + 1Ck8M SP to isolated porcine ophthalmic arteries precontracted by 10e5 M PGF,, in vitro. NS: non significant, Student’s t test. 7 5 n 2 9.
highly significant (p < 0.0005). At this point, SPinduced relaxation was 6.5 k 2.1%, showing that the combination of SP + CGRP induced a relaxation which was almost twice as large as might be expected from the additive effects of SP and CGRP alone.
Discussion
The dose-response relaxations are to some extent in accordance with neuropeptide dilator activity reported for other vascular beds in the head. CGRP is considered the most potent vasodilator so far (13). By comparison, SP had much less vasodilator activ-
25 zo-
.qJ_
CGRP
+-
9
+
SP+CGRP
15 s ‘J 3 m
lo-
:
5o-
0
10
20
30
40
50
60
time (set)
Fig. 3
Relaxation Curves obtained by adding 10” M CGRP, 1t8 M SP (n = 9) and 1W M CGRP + lo-8 M SP (n = 8) to isolated porcine ophthalmic arteries pre-contracted by lO_’ M PGF,, in vitro. Statistical analysis of the differences between the CGRP and SP + CGRP relaxations: * p < 0.001. 0 p < 0.01. t p < 0.05. Values represent the mean k S.E.M.
ity, which is in contrast to its activity e.g. in human temporal arteries (13). SP is also a potent relaxation factor in human cerebral arteries (14). The reasons for the relatively weak relaxations induced by SP in the porcine ophthalmic artery are unknown. Further studies are required in order to verify whether this represents a trait specific for this arterial bed. The results show that, in the porcine ophthalmic SP-induced relaxation artery, the maximum occurred with a concentration of IO-* M. Since the same amount of CGRP provoked a large relaxation, this concentration has been chosen for the experiments over time. The computerized system accurately records the exact time peptides were added, which makes the observation of time parameters possible. By using consecutive slices of the same artery in parallel experiments, differences in response due to anatomical traits such as artery wall thickness or individual variations have been theoretically reduced. The results obtained suggest that modifications in the rate of peptide response may represent important additional information in vasoactive neurotransmitter studies. Had this study been limited to determination of the maximum effects of the reactions, no differences between CGRP- and SP + CGRP-induced relaxations would have been detected. Differences between the induced relaxations were observed mainly during the first minutes of the relaxation curve. Such data may thus provide important information related to the modulation of vascular responses over time. SP induced a more rapid, but short relaxation in comparison with CGRP. Although speculative, it is possible that the type of vascular response following antidromic trigeminal stimulation could be dependent on the SPCGRP ratio. It is quite possible that peptides are liberated according to the circumstances (for instance, frequency of stimulation). Thus, in this arterial bed, SP could be the trigeminal quick, but short-lasting vasodilator, whereas CGRP could be the inducer of slow and long-lasting relaxations, a combination providing both a rapid and long-lasting response. The intensity, rate, and duration of vascular responses would thus be dependent upon the SPKGRP (and/or other transmitters) concentration ratio. It is possible that differences in such ratios could explain, for instance, why cluster headache
SP AUGMENTS
THE RATE OF CGRP IN PORCINE OPHTHALMIC
attacks are more long-lasting than those of chronic paroxysmal hemicrania, if such peptides are involved in the pathophysiology of headache. However, the role for this putative synergism between SP and CGRP has stili to be determined. It is concluded that, in porcine ophthalmic arteries in vitro, the rate of relaxation induced by 10e8M CGRP together with 1O-8M SP is higher than that induced by 1O-8M CGRP alone. A SP-CGRP synergism is suggested, since the relaxation induced by a SP + CGRP combination was greater than what would be expected from the effects obtained with these peptides individually. In this artery, the SPinduced relaxations were quicker and shorter when relaxations were induced with CGRP. However, such effects were present only initially, before the CGRP maximum effect was reached. The interactions between different neurotransmitters may be important in cerebral blood flow regulation concerning the timing of the reflex responses and duration of the effects. From the pathophysiological point of view, such interactions may be considered of importance in cerebrovascular disorders or headache. Acknowledgements MBV is indebted to the Brazilian Research Council, CNPq, for a grant during this work (no. 200400/89.4). The authors wish to thank the Norwegian National Health Association (Nasjonalforeningen. Det Norske ad for Hjerte- og Karsykdommer) for financial support, Professor A. Brubakk for permission to use porcine material and the animal quarters at the University Hospital for their cooperation.
References 1. Beal, M. F. and Martin, J. B. (1986). Neuropeptides in neurological disease. Annals of Neurology 20: 247-565. 2. Uddman, R. and Edvinsson, L. (1989). Neuropeptides in the cerebral circulation. Cerebrovascular and Brain Metabolism Reviews 1: 230-252.
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141
3. Hardebo, J. E. (1984). The involvement of trigeminal substance P neurons in cluster headache. An hypothesis. Headache 24: 294-304. 4. Moskowitz, M. A. (1984). The neurobiology ofvascularhead pain. Annals ofNeurology 16: 157-168. 5. Hiikfelt, T., Johansson, O., Ljungdahl, A.. Lundberg, J. M. and Schultzberg, M. (1980). Peptidergic neurones. Nature 284: 515-521. 6. Kuwayama, Y., Terenghi, G.. Polak, J. M., Trojanowski. J. Q. and Stone. R. A. (1987). A quantitative correlation of substance P-, calcitonin gene-related peptide- and colecystokinin-like immunoreactivity with retrograde labelled trigeminal ganglion cells innervating the eye. Brain Research 405: 220-226. 7. Wahlestedt, C.. Beding, B., Ekman. R.. Oksala, 0.. Stjernschantz. J. and Hhkanson, R. ( 1986). Calcitonin generelated peptide in the eye: release by sensory nerve stimulation and effects associated with neurogenic infl;immation. Regulatory Peptides 16: 107-l 15. 8. Brain. S. D. and Williams, T. J. (1988). Substance P regulates the vasodilator activity of calcitonin gene-related peptide. Nature 335: 73-75. 9. Wiesenfeld-Hallin, Z., Hiikfelt, T., Lundberg, J. M., Forssman, W. G., Reinecke, M., Tschopp, F. A. and Fischer. J. A. (1984). Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavior responses of the rat. Neuroscience Letters 52: 199-204. 10. Smullin, D. H., Skilling, S. R. and Larson, A. A. (1990). Interactions between substance P, calcitonin gene-related peptide, taurine and excitatory amino acids in the spinal cord. Pain 42: 93-101. 11. Le Greves, P., Nyberg, F., Terenius, L. and Hbkfelt, T. (1985). Calcitonin gene-related peptide is a potent inhibitor of substance P degradation. European Journal of Pharmacology 115: 309-311. 12. Manzoni, C. G., Terzano, M. G., Bono. G., Micieli, G.. Martucci, N. andNappi, G. (1983). Cluster headache-clinical findings in 180 patients. Cephalalgia 3: 2 l-30. 13. Jansen, I.. Uddman, R., Hocherman, M., Ekman, R., Jensen, K.. Olesen, J.. Stiernholm, P. and Edvinsson. L. (1986). Localization and effects of neuropeptide Y. vasoactive intestinal polypeptide, substance P, and calcitonin generelated peptide in human temporal arteries. Annals of Neurology 20: 496-50 1. 14. Edvinsson. L., Ekman, R., Jansen, I., Ottosson, A. and Uddman, R. (1987). Peptide-containing nerve fibers in human cerebral arteries: Immunocytochemistry. radioimmunoassay and in vitro pharmacology. Annals ofNeurology 21: 431-437.
Substance P Augments the Rate of Vasodilation Induced by Calcitonin Gene-related Peptide in Porcine Ophthalmic Artery In Vitro M. B. VINCENT*, L. R. WHITE, T. ELSAS, G. QVIGSTAD and 0. SJAASTAD Department of Neurology, Trondheim University Hospital, Norway. *Department of Neurology, University Hospital, Federal University of Rio de Janeiro, 21949 Rio de Janeiro, Brazil (Reprint requests to MBV)
Abstract - Peptides may function as neurotransmitters liberated antidromically by sensory nerve fibres, provoking vascular responses having potential importance in some neurological disorders. Dose-response relaxation curves induced by substance P (SP) and calcitonin gene related peptide (CGRP) have been studied in porcine ophthalmic arteries in vitro. Both peptides induced vasodilation when tested separately (CGRP >> SP). Because of the putative interactions between such peptides in this vascular territory, a computerised system was also used for analysing over time the response to a single addition of either 10q M CGRP, lo4 M SP or a combination of 10q M SP + lOa M CGRP. SP did not augment the maximum relaxation induced by CGRP alone, but increased significantly the rate of relaxation during the initial phase of the response. The effect induced by the SP+CGRP combination was stronger than the sum of the individual SP and CGRP-induced relaxations during the first 4 min of the response, which suggests a SP-CGRP synergism in this artery.
Introduction Neuropeptides have been implicated in the pathophysiology of different neurological diseases (1). They also seem to be important in cerebral flow regulation (2) and have been suggested to be involved in headaches (3,4). Several neuropeptides may coexist with ‘classical’ neurotransmitters within the Date received 2 December 199 1 Date accepted 5 February 1992 Address for correspondence: Department of Neurology, Trondheim University Hospital, N-7006 Trondheim, Norway.
same fibres (5), possibly having intricate interactions not yet understood. The undecapeptide substance P (SP) is known to be present in trigeminal sensory fibres, as well as calcitonin gene-related peptide (CGRP), a 37 aminoacid peptide encoded by the calcitonin gene (6). SP-CGRP interactions have already been described in several studies. Intravitreal injections of CGRP in rabbits did not change the pupillar diameter, but the miosis produced by SP and CGRP in combination was greater than that produced by SP alone (7). The long lasting CGRP-induced vasodila137
138 tor response observed after an intradermal injection in humans was much shorter if SP was also injected, a phenomenon probably involving mast cell activation (8). CGRP potentiated the SP-induced biting and scratching behaviour when injected intrathetally in rats, whereas the administration of CGRP alone produced no behavioural changes (9). CGRP alone produced no release of excitatory aminoacids if infused into the rat dorsal spinal cord, but potentiated the apparent SP-induced release of taurine (10). CGRP was also shown to inhibit SP degradation by acting on an SP endopeptidase isolated from human CSF (11). The ocular and forehead circulation may play an important role in the pathophysiology of headaches. For instance, conjunctival injection, vasodilation, and lacrimation are known to occur in cluster headache (12). Both SP and CGRP are known to induce vasodilation in different arteries in the head (2), but the combined effects of SP and CGRP in this vascular bed, which may occur in vivo following sensory fibre activation, are unknown. The objective of this study was to investigate the vasodilation induced by CGRP and SP, separately and combined, in porcine ophthalmic artery in vitro.
Materials and Methods The entire orbital contents were removed from pigs anaesthetised with a-choralose and killed with a lethal dose of pentobarbital i.v., placed in iced, 02bubbled buffer solution (NaCl 119, KC1 4.6, CaCL 1.5, MgCh 1.2, NaHC03 10, NaH2P04 1.2, glucose 11. Values in 10” M) and kept at 4°C for 15 17 h. Retroocular segments of the ophthalmic artery were gently dissected with the aid of a stereomicroscope. Artery slices (1 to 3 mm) were mounted on two Lshaped steel holders (0.1 mm diameter), where one of the holders was connected to a Grass FT03C force-displacement transducer. For recording the arterial constriction forces, the transducers were linked to an Analog Digital Instruments MacLab Analog-Digital convertor through a Transbridge TBM4 preamplifier (World Precision Instruments, Inc, New Haven CT, USA). The digitalized data were analysed by MacLab’s Chart/4 software in an Apple Macintosh SE 1140 computer. The holders were immersed in 5 ml buffer solution (37’C, pH
NEUROPEPTIDES
7.4) which was continuously bubbled with a 5% C02, 95% O2 mixture. A passive tension of 4-5 mN was applied to the segments followed by an equilibration period of 1 - 1.5 h, until the tension was stable. The buffer solution was changed every 10 min throughout the equilibration period. The arteries were first contracted by 10-j M PGF2, and when a stable reaction was reached, the relaxation induced by the peptides was studied. PGF2, at a concentration of 10-j M was chosen as the best pre-contractor agent, since it proved to induce stable and long lasting contractions in this artery, suitable for performing dose-response experiments (data not shown). In order to obtain dose response relaxation curves, SP (acetate salt) or a-CGRP (human, synthetic) were added cumulatively in logarithmically increasing concentrations (SP: IO-loto 10” M. CGRP: lo-lo to lo-’ M). For studying the induced relaxation over 1 h, the time peptides were added was accurately recorded by the computer, and the relaxing response continuously registered throughout the following 60 min. SP experiments were terminated after 30 min since the SP-induced relaxations were short-lasting. The computer recorded the constriction forces every 6 s. Average curves representing the peptides activity over time were obtained from the digitalized values stored on the computer. The experiments were performed in parallel baths using consecutive slices of the same artery. A new animal was used for each set of experiments. The data are presented as the mean + S.E.M. Statistical analysis was carried out with Student’s t-test. Values of p < 0.05 were considered significant. All substances were added in 50 pl aliquots. Drugs were purchased from Sigma Chemical Co., St. Louis, MO, USA and were diluted in 0.9% NaCl containing 10” M ascorbic acid.
Results Dose-response experiments Figure 1 shows the relaxations obtained by adding cumulatively SP (1 O-loto 10” M) or CGRP (1 VI0 to 1V7 M) to isolated porcine ophthalmic arteries precontracted with 1V5 M PGFza. At lo-lo M, both peptides induced a relaxation of around 5%, while at 1O-9M, the CGRP-induced relaxation was higher
SP AUGMENTS
THE RATE OF CGRP IN PORCINE
OPHTHALMIC
ARTERY
139
IN VITRO
80 c .o iii
80
3 E
40
s
20
-10
-9
-8
-7
-8
-5
[peptide] (log M) Fig. 1 Relaxations induced by adding cumulatively, CGRP (lWO to lO_’M) or SP (lo-l0 to 1k5 M) to isolated porcine ophthalmic arteries pre-contracted by 1tYsM PGF*, in vitro. At 1W0 and lo+ M: no significant difference. At 10-s M and 1W M: p < 0.00001. Values represent the mean k S.E.M., n = 9.
than that induced by SP, but the difference was not statistically significant. However, at lo-* and lo-’ M, CGRP induced a relaxation significantly higher than SP (p < 0.00001). CGRP was not tested at concentrations higher than lo-’ M. SP was tested up to a concentration of 1O-5M, but induced its maximum effect at l@* M. Relaxation studies over time Figure 2a shows the relaxation curves induced by the addition of lo-* M CGRP, lo-8 M SP, or 1O-8M CGRP together with lo4 M SP, to porcine ophthalmic arteries pre-contracted by 1e5 M PGF2,. The reaction was studied continuously for approximately 1 h (CGRP) or 30 min (SP). The maximum relaxation induced by SP (around 15%), occurred on average after 3.0 + 0.6 min, and by CGRP (around 85%), after ca. 11.Of 0.7 min (p < 0.0000 1, Fig. 2a). When comparing the effects of CGRP alone and CGRP+SP, both curves were found to achieve maximum relaxation (around 85%) after about 10 min. Statistically, no difference was found in the values for the maximum effect (I,,), nor in the time taken to achieve I,,, (Table). Despite the tendency for a longer-lasting relaxation with the combination of peptides, no significant differences were found either in the time necessary to achieve a 50% recovery from the maximum effect (R,,), nor in the effects observed after 1 h (Table). In Figure 2b, these data are shown in greater detail to highlight the reaction during the first 7 min. A line
0
1
2
3
1
5
B
7
time (min)
Fig. 2 Average relaxation curves obtained by adding 1O-8M CGRP(n=8),1~8MP(n=7)and10-*MCGRP+10_8MSP (n = 7) to isolated porcine ophthalmic arteries pre-contracted by 1O-5M PGF2, in vitro. Fig. 2a: reaction durina 1h. S&es on the curves are electrical at-&acts and do not represent ;asoactivity. Fig. 2b: reaction during the first 7 mm. The dotted line represents the sum of the individual SP and CGRP reactions.
representing the sum of the SP effect plus the CGRP effect has also been included. This figure shows that the relaxation induced by the SP + CGRP combination was higher than the sum of each reaction individually during the first 4 min. The relaxation induced by SP declined after 3 min, whereas the CGRP and the CGRP + SP effects continued to increase. It is clear (Figs. 2b and 3) that there was an apparent difference in the initial rate of relaxation following peptide addition. Analysis of this early reaction (Fig. 3) showed that as early as 6 s after addition of peptides, the relaxation induced by SP together with CGRP was already stronger than that induced by CGRP alone, but the difference became significant (p < O.OS), first after 18 s. By 1 min the SP + CGRP combination induced a relaxation of 21.5 + 3.5%, with a corresponding relaxation of CGRP alone of only 4.7 f 1.4%. This difference was
140
NEUROPEPTIDES
Table
Time parameters SP+CGRP
CGRP
84.90+5.52 10.30 f 1.75 26.12 f 6.16 25.53 k 9.98
La, (%I tI,, (min) R0 (mm) E, ,, (%)
P
86.99f 5.20 11.02 f 0.75 24.47 f 4.2 1 20.59 f 5.76
NS NS NS NS
Maximum effect (I_, % relaxation), time necessary for reaching the maximum effect (tI,,,,, min), time necessary for a 50% recovery after maximum effect (&,, min) and effect present after 1 h (E, ,,, % relaxation) after adding 1W M CGRP and 10e8M CGRP + 1Ck8M SP to isolated porcine ophthalmic arteries precontracted by 10e5 M PGF,, in vitro. NS: non significant, Student’s t test. 7 5 n 2 9.
highly significant (p < 0.0005). At this point, SPinduced relaxation was 6.5 k 2.1%, showing that the combination of SP + CGRP induced a relaxation which was almost twice as large as might be expected from the additive effects of SP and CGRP alone.
Discussion
The dose-response relaxations are to some extent in accordance with neuropeptide dilator activity reported for other vascular beds in the head. CGRP is considered the most potent vasodilator so far (13). By comparison, SP had much less vasodilator activ-
25 zo-
.qJ_
CGRP
+-
9
+
SP+CGRP
15 s ‘J 3 m
lo-
:
5o-
0
10
20
30
40
50
60
time (set)
Fig. 3
Relaxation Curves obtained by adding 10” M CGRP, 1t8 M SP (n = 9) and 1W M CGRP + lo-8 M SP (n = 8) to isolated porcine ophthalmic arteries pre-contracted by lO_’ M PGF,, in vitro. Statistical analysis of the differences between the CGRP and SP + CGRP relaxations: * p < 0.001. 0 p < 0.01. t p < 0.05. Values represent the mean k S.E.M.
ity, which is in contrast to its activity e.g. in human temporal arteries (13). SP is also a potent relaxation factor in human cerebral arteries (14). The reasons for the relatively weak relaxations induced by SP in the porcine ophthalmic artery are unknown. Further studies are required in order to verify whether this represents a trait specific for this arterial bed. The results show that, in the porcine ophthalmic SP-induced relaxation artery, the maximum occurred with a concentration of IO-* M. Since the same amount of CGRP provoked a large relaxation, this concentration has been chosen for the experiments over time. The computerized system accurately records the exact time peptides were added, which makes the observation of time parameters possible. By using consecutive slices of the same artery in parallel experiments, differences in response due to anatomical traits such as artery wall thickness or individual variations have been theoretically reduced. The results obtained suggest that modifications in the rate of peptide response may represent important additional information in vasoactive neurotransmitter studies. Had this study been limited to determination of the maximum effects of the reactions, no differences between CGRP- and SP + CGRP-induced relaxations would have been detected. Differences between the induced relaxations were observed mainly during the first minutes of the relaxation curve. Such data may thus provide important information related to the modulation of vascular responses over time. SP induced a more rapid, but short relaxation in comparison with CGRP. Although speculative, it is possible that the type of vascular response following antidromic trigeminal stimulation could be dependent on the SPCGRP ratio. It is quite possible that peptides are liberated according to the circumstances (for instance, frequency of stimulation). Thus, in this arterial bed, SP could be the trigeminal quick, but short-lasting vasodilator, whereas CGRP could be the inducer of slow and long-lasting relaxations, a combination providing both a rapid and long-lasting response. The intensity, rate, and duration of vascular responses would thus be dependent upon the SPKGRP (and/or other transmitters) concentration ratio. It is possible that differences in such ratios could explain, for instance, why cluster headache
SP AUGMENTS
THE RATE OF CGRP IN PORCINE OPHTHALMIC
attacks are more long-lasting than those of chronic paroxysmal hemicrania, if such peptides are involved in the pathophysiology of headache. However, the role for this putative synergism between SP and CGRP has stili to be determined. It is concluded that, in porcine ophthalmic arteries in vitro, the rate of relaxation induced by 10e8M CGRP together with 1O-8M SP is higher than that induced by 1O-8M CGRP alone. A SP-CGRP synergism is suggested, since the relaxation induced by a SP + CGRP combination was greater than what would be expected from the effects obtained with these peptides individually. In this artery, the SPinduced relaxations were quicker and shorter when relaxations were induced with CGRP. However, such effects were present only initially, before the CGRP maximum effect was reached. The interactions between different neurotransmitters may be important in cerebral blood flow regulation concerning the timing of the reflex responses and duration of the effects. From the pathophysiological point of view, such interactions may be considered of importance in cerebrovascular disorders or headache. Acknowledgements MBV is indebted to the Brazilian Research Council, CNPq, for a grant during this work (no. 200400/89.4). The authors wish to thank the Norwegian National Health Association (Nasjonalforeningen. Det Norske ad for Hjerte- og Karsykdommer) for financial support, Professor A. Brubakk for permission to use porcine material and the animal quarters at the University Hospital for their cooperation.
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